Anti-ifnar1 antibodies with lower ligand fc affinity

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. What is presented is a monoclonal anti-IFNAR1 antibodies with L234F, L235E and P331S Fc mutations of human IgG1 possessing a lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors as compared to a non-modified antibody. There are described the recovered nucleic acid providing expression of the above antibody containing a nucleotide sequence coding the antibody, and a pharmaceutical composition based on the above antibody.

EFFECT: using the invention provides the antibody possessing the lower affinity to Fcgamma RI, Fcgamma RIIIA and c1q receptors that provides reducing the undesired effector functions in treating chronic inflammation and autoimmune conditions.

9 cl, 34 dwg, 7 tbl, 36 ex

 

According to this application claims priority under section 35 U. S. C. §119(e) on the basis of the following provisional patent applications U.S.: US 61/006962, filed February 8, 2008, 61/034618, filed March 7, 2007, and 61/049970, filed may 2, 2008, the essence of which is incorporated into this description by reference.

The technical field to which the invention relates

The present invention relates to the selected antibody and compositions that are specific in relation to receptor 1 interferon alpha (interferon alpha receptor 1 - IFNAR1), with a reduced affinity against Fc ligands. The present invention also relates to nucleic acids encoding such antibodies, complementary nucleic acids, vectors, cells-owners, and methods for their preparation and use, including therapeutic compositions, formulations, methods of administration and device.

The level of technology

Interferons

Interferons (IFN) type I (IFNα, IFNβ, IFNω, IFNτ) represent a family of structurally related cytokines having antiviral, antitumor and immunomodulatory effects (Hardy et Blood 97, 2001, P. 473; Cutrone and Langer J. Biol. Chem. 276, 2001, p. 17140). The locus of the human IFNα includes two subfamilies. The first subfamily consists of 14 nonallelic genes and 4 pseudogenes having at least 80% homology. The second subfamily, αII or omega (ω), includes 5 pseudo�new and 1 functional gene having 70% homology with IFNα genes (Weissmann and Weber, Prog. Nucl. Acid Res. Mol. Biol., 33, 1986, cc.251-300). The IFNα subtypes have different specific actions, but the same biological spectrum (Streuli. Proc. Natl. Acad. Sci. USA 78, 1981, p. 2848) and the same cell receptor (Agnet M. and others in kN.: "Interferon 5", 1983, ed. by I. Gresser, publ Academic Press, London, cc.1-22). Subtypes of interferon alpha denote as follows: IFNα 1, 2A, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21.

Interferon β (IFNβ) encodes one gene, which is approximately 50% homologous to genes IFNα.

Interferon-γ secreted by activated lymphocytes, does not show any homology with alpha/beta interferons and does not interact with their receptor.

Receptors interferon

All type I interferons man bind to a receptor on the cell surface (receptor IFN alpha, IFNAR) consisting of two transmembrane proteins IFNAR1 and IFNAR2 (Uze et Cell 60, 1990, p. 225; Novick, etc. Cell 77, 1994, p. 391). IFNAR1 protein essential for high binding affinity and differential specificity of the IFNAR complex (Cutrone et J. Bio Chem 276(20), 2001, cc.17140-17148). Although functional differences for each subtype of type I IFN was not detected, suggesting that each subtype may exhibit different interactions with components of the receptor IFNAR, leading to a possible difference between the results for the transmission of signals (Cook and others, J. Biol. Chem. 271, 1996, pp. 13448). In particular, and�distance using mutant forms of proteins IFNAR1 and IFNAR2, confirm that the signals alpha and beta interferons have different transmitted via the receptor through differential interaction with relevant circuits (Lewerenz et J. Mol. Biol. 282, 1998, p. 585).

Functions of interferons

Early functional studies of type I interferons have focused on innate defense against viral infections (Haller et J. Exp. Med. 154, 1981, p. 199; Lindenmann, etc. Methods Enzymol. 78, 1981, p. 181). However, in later studies examined the type I interferons as a potent immunoregulatory cytokine in the formation of the adaptive immune response. Especially it was shown that type I interferons facilitate the differentiation is not subjected to any impact of T cells on metabolic pathway Th1 (Brinkmann et J. Exp. Med. 178, 1993, p. 1655) to enhance antibody production (Finkelman and others, J. Exp. Med. 174, 1991, p. 1179) and support for the functional operation and survival of T-memory cells (Santini et J. Exp. Med. 191, 2000, p. 1777; Tough, etc. Science 272, 1996, p. 1947).

Some groups of researchers in previous studies have suggested that IFNα may enhance the maturation or activation dendrocygna cells (DC) (Santini et (2000) J. Exp. Med. 191:1777; Luft et J. Immunol. 161, 1998, p. 1947; Luft et Int. Immunol. 14, 2002, p. 367; Radvanyi, etc. Scand. J. Immunol. 50, 1999, p. 499). In addition, increased expression of type I interferons description�on with numerous autoimmune diseases (Foulis, etc. Lancet 2, 1987, p. 1423; Hooks, etc. Arthritis Rheum. 25, 1982, p. 396; Hertzog et Clin. Immunol. Immunopathol. 48, 1988, p. 192; Hopkins and Meager Clin. Exp. Immunol. 73, 1988, p. 88; Arvin and Miller Arthritis Rheum. 27, 1984, p. 582). Most related studied examples is insulin-dependent diabetes mellitus (IDDM) (Foulis (1987)) and systemic lupus erythematosus (SLE) (Hooks (1982)), which is associated with increased IFNα levels, and rheumatoid arthritis (PA) (Hertzog (1988), Hopkins and Meager (1988), Arvin and Miller (1984)), in which IFNβ may play a more significant role.

In addition, reported that administration of interferon α exacerbate underlying disease in patients with psoriasis and multiple sclerosis, as well as the induction of SLE-like syndrome in patients who previously did not have autoimmune diseases. It was also found that interferon-α induces glomerulonephritis in normal mice and to accelerate the start of spontaneous autoimmune disease in mice NZB/W. in addition, it was found that IFNα therapy may in some cases lead to undesirable side effects, including fever and neurological disorders. Consequently, there are pathological situations in which inhibition of the action of type I IFN may be useful for the patient, and there is a need for agents effective for inhibiting the action of type I IFN.

Effector functions of antibodies

The Fc region of an antibody interacts with a number of ligang�s (also called the "Fc ligands", which include, but which the list is not limited to, agents specifically binding to the Fc region of antibodies, for example, Fc receptors and C1q), including Fc receptors and C1q, ensuring proper functioning of vital signs, called effector functions. Fc receptors mediate communication between antibodies and the cellular processes of the immune system (Raghavan et, Annu Rev Cell Dev Biol 12, 1996, cc.181-220; Ravetch et, Annu Rev Immunol 19, 2001, cc.275-290). In humans this family of proteins include: FcγRI (CD64), including isoforms FcγRIA, FcγRIB and FcγRIC; FcγRII (CD32), including isoforms FcγRIIA, FcγRIIB and FcγRIIC; and FcγRIII (CD16), including isoforms FcγRIIIA and FcγRIIB (Jefferis et, Immunol Lett 82, 2002, cc.57-65). These receptors typically have an extracellular domain that mediates binding to Fc, the area of membrane binding and intracellular domain, which may mediate some signaling in the cell. These receptors are expressed in various immune cells, including monocytes, macrophages, neutrophils, dendrobiinae cells, eosinophils, mast cells, platelets, b cells, large granular lymphocytes, Langerhans cells, natural killer cells (NK) and T cells. The formation of the complex Fc/FcγR recruits these effector cells to sites of bound antigen, typically resulting in the transmission of signals inside the cells and important subsequent immune response�m, for example, to the release of inflammatory mediators, activation of b-cells, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector function is an important mechanism by which antibodies upset the target cell. Mediated cell reaction in which nonspecific cytotoxic cells expressing FcγR, recognize bound antibody on the target cells and then cause lysis of the target cells is called antibody-dependent cell-mediated cytotoxicity (antibody dependent cellular cytotoxicity - ADCC) (Raghavan et, Annu Rev Cell Dev Biol 12, 1996, cc.181-220; Ghetie et, Annu Rev Immunol 18, 2000, cc.739-766; Ravetch, etc., Annu Rev Immunol 19, 2001, cc.275-290). Mediated cell reaction in which nonspecific cytotoxic cells expressing FcγR, recognize bound antibody on a target cell and then cause phagocytosis of the target cells is called antibody-dependent cell-mediated phagocytosis (antibody dependent cell-also been other ideas where phagocytosis - ADCP). In addition, the overlapping site in the Fc region of the molecule also controls the activation of cell-independent cytotoxic functions mediated by complement, also called complement-dependent cytotoxicity (complement dependent cytotoxicity CDC).

Different types of human FcγR

The FcγR receptors of man is divided into three time�'s classes: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Receptor FcγRI is a receptor with high affinity (Ka: 10-8-10-9M-1and associates and immune complexes and Monomeric IgG molecules, although Fc receptors FcγRII and FcγRIII inhibit the low affinity (<10-7M-1and 2-3×10-7respectively) (J. E. Gessner, etc., Annn Hematology 76, 1998, cc.231-248). The transmission of signals via FcγR occurs either via immunoreceptor tyrosine-based activating motif (immunoreceptor tyrosine-based activation motif ITAM), or through immunoreceptor tyrosine-based inhibitory motif (immunoreceptor tyrosine-based inhibitory motif ITIM) for all transmembrane receptors (Presta, Adv Drug Deli Rev 58, 2006, cc.640-656).

Extracellular glycoprotein FcγRI mass of 72 kDa is expressed predominantly on myeloid cells, e.g., monocytes, progenitor cells macrophages CD4+ and may cause responses in the form of ADCC, endocytosis and phagocytosis (Siberil et J Immunol Lett 106, 2006, cc.111-118).

Receptors group FcγRII mass of 40 kDa (A, b and C isoforms) exhibit extracellular domains, but do not contain the active domains of signal transduction. These receptors propagate signals through phosphorylation of the cytoplasmic tail domain (Amigorena S., etc., Science. 256, 1992, cc.1808-1812). The FcγRIIA receptor mainly expressed on monocytes, macrophages, neutrophils and platelets, although the FcγRIIC receptor was identificere�EN only on NK cells. It was found that these two receptor initiate ADCC, endocytosis, phagocytosis, and the release of a mediator of inflammation (Cassel, etc., Mol Immunol 30, 1993, cc.451-460). In contrast, FcγRIIB receptors (types B1 and B2) expressed on b cells, mast cells, basophils, monocytes, macrophages and dendrometric cells, and also found that they reduce the immune response triggered by isoforms A and C.

The FcγRIIIA receptor mass of 50 kDa, expressed on NK cells, monocytes, macrophages and a subset of T-lymphocytes, where it activates ADCC, phagocytosis, endocytosis and release of cytokine (Gessner and others). Isoform FcγRIIIB is glycosylphosphatidylinositol (glycosyl-called phosphatidylinositol - GPI) anchored peripheral membrane protein involved in degranulation and the development of reactive intermediates of oxygen (J. E. Salmon, etc,. J Clin Inves 95, 1995, cc.2877-2785).

IgG molecules also exhibit different isotypes specificity receptor FcγR. The IgG3 molecule strongly associated with all FcγR isoforms. Isoforms IgG1, the most prevalent isoform in the blood, is associated with all FcγR receptors, although with lower affinity in respect of the isoforms FcγRIIA/B. IgG4 is an intermediate binding to FcγRI and weak binding to FcγRIIB. Ultimately IgG2 binds weakly with only one allelic form of FcγRIIA (FcγRIIA-H131) (Siberil et, J Immunol Lett 106, 2006, cc.111-118).

Complement

Inflammatory complement cascade is part of the innate immune response and is the key in relation to the individual's ability to contain the infection. Another important Fc ligand is a protein of complement C1q. Fc by binding to C1q, mediates a process called complement-dependent cytotoxicity (complement dependent cytotoxicity CDC) (on this topic, see the review of Ward, etc., Ther Immunol 2, 1995, cc.77-94). Able to bind Clq six antibodies, although linking the two IgG is sufficient to activate the complement cascade. C1q forms a complex with the serine proteases C1r and C1s to form the C1 complex metabolic pathway of complement.

Region and IgG amino acid residues involved in the binding of FcγR

Conducted a thorough study of the mapping of the binding sites of human IgG with different FcγR receptors. These studies, based on genetically modified IgG molecules revealed a short continuous sections of amino acid residues (234-238) N-terminal portion of the CH2 domain, as directly involved in binding to all FcγR receptors. In addition, residues 268, 297, 327 and 329 may affect the binding of a subgroup of the FcγR. In addition, multiple residues, localized to domains CH2 and CH3, also affect the binding to FcγR (Canfield, S. M. and others., J Exp Med 173, 1991, cc.1483-1491; M. S. Chappel, etc. Proc Nat Acad Sci USA 888, 1991, cc.9036-9040; J. Gergely, etc., FASEB J 4, 990, cc.3275-3283).

Toxicity associated with therapeutic antibodies

In many cases, the binding and stimulation of effector functions mediated by the Fc region of immunoglobulin, is useful, but in some cases it may be more useful to reduce or eliminate effector function. This is especially true for those antibodies that are designed to deliver drugs (e.g., toxins and isotopes) to target cells, when the Fc/FcγR-mediated effector functions contribute to healthy immune cells into contact with a deadly cargo, resulting in depletion of normal lymphoid tissue along with the target cells (Hutchins et, PNAS USA 92, 1995, cc.11980-11984; White and others, Annu Rev Med 52, 2001, cc.125-145). In these cases, the use of antibodies that are weakly recruited in the presence of complement or effector cells, can have a huge advantage (see, e.g., Wu, etc., Cell Immunol 200, 2000, cc.16-26; Shields, etc., J. Biol Chem 276, 2001, cc.6591-6604; US 6194551; US 5885573 and PCT publication WO 04/029207).

In other cases, for example, if the goal is to block the interaction of a widely expressed receptor with its closest ligand, it may be useful to reduce or eliminate all of the effector functions of the antibody to reduce unwanted toxicity. In addition, if therapeutic antibody shows promiscuous binding to several fabric� person it may be appropriate to limit the targeting of effector functions on a diverse set of tissues to limit toxicity. Although some well-known subclasses with immunoglobulin human who have lost specific effector functions, unknown natural immunoglobulins, lost effector function. Another approach might be designing or insertion mutations in relation to the fundamentally important residues in the Fc region responsible for effector function. See, for example, PCT publication WO2006076594, WO199958572, US20060134709, WO2006047350, WO2006053301 and US 5624821, each of which is included in the present invention in reference to its essence.

The use of monoclonal antibodies for the treatment of many diseases is well studied. With countless effector functions of the antibody may induce, one of the necessary conditions for therapeutic antibodies is that they specifically target to the desired target. For example, but this example is not restrictive, the specificity of the target tissue is analyzed by determining immunohistochemistry (IHC) of the investigated tissue. It is important that the drug is associated only with fabrics that contain the studied target. Disorder such actions may lead to increased toxicity of therapeutic antibodies due to the inadequate �Stivali effector function, caused by the website not being the target. If the effector function can be reduced or removed, the danger of widespread binding of a therapeutic agent can be avoided. Given the above, there is an unsolved problem for the development of antibodies with reduced or removed by affinity in relation to at least one Fc ligand, is responsible for the action of effector functions. Such antibodies can be particularly useful for use in the treatment of chronic inflammation and autoimmune conditions.

In the present invention citation and discussion of the links should not be interpreted as an assumption that is prior to the present invention.

Brief description of figures

To illustrate the present invention in the form of the figures presented certain embodiments of the present invention. However, the present invention is not limited to the exact description of manipulation and instrumentation of the variants of the present invention, which are displayed in the figures.

Fig.1A. The alignment of the nucleic acid sequence (SEQ ID No:7) and the alignment of the amino acid sequence (SEQ ID No:8) 3F11 VH CDR regions that are underlined above.

Fig.1B. The alignment of the nucleic acid sequence (SEQ ID No:9) and amino acid follower�spine (SEQ ID No:10) 3F11 VK CDR regions, underlined above.

Fig.2A. The alignment of the nucleic acid sequence (SEQ ID No:17) and the alignment of the amino acid sequence (SEQ ID No:18) 4G5 VH CDR regions that are underlined above.

Fig.2B. The alignment of the nucleic acid sequence (SEQ ID No:19) and the alignment of the amino acid sequence (SEQ ID No:20) 4G5 VK with areas of a CDR are underlined above.

Fig.3A. The alignment of the nucleic acid sequence (SEQ ID No:27) and the alignment of the amino acid sequence (SEQ ID No:28) 11E2 VH CDR regions that are underlined above.

Fig.3B. The alignment of the nucleic acid sequence (SEQ ID No:29) and the alignment of the amino acid sequence (SEQ ID No:30) 11E2 VK with areas of a CDR are underlined above.

Fig.4A. The alignment of the nucleic acid sequence (SEQ ID No:37) and the alignment of the amino acid sequence (SEQ ID No:38) 9D4 VH CDR regions that are underlined above.

Fig.4B. The alignment of the nucleic acid sequence (SEQ ID No:39) and the alignment of the amino acid sequence (SEQ ID No:40) 9D4 VK with areas of a CDR are underlined above.

Fig.5. Alignment of amino acid sequences of the constant regions of the heavy chain for 9D4. Arrows indicate amino acid substitution (remodification� modified) to improve stability and reduced affinity in relation to at least one Fc ligand.

Fig.6A. Profile immunohistochemical staining of tissue główna brain treated with various anti-IFNAR1 antibodies. Antibody 9D4 demonstrates the low profile of staining after incubation with tissue in the human brain compared to the antibody 4G5 and MDX-1333.

Fig.6B. Profile immunohistochemical staining of human monocytes treated with various anti-IFNAR1 antibodies. As a positive control, a variety of anti-IFNAR1 antibodies can be tested for reactivity against human monocytes.

Fig.7. Anti-IFNAR1 antibody 9D4 inhibits IFNα signal transmission according to a study of the activation of STAT-based cells. Treatment with antibody 9D4 inhibits STAT1/3/4 tyrosine phosphorylation in response to stimulation with interferon alpha, which was established by the method of Western blotting with commercially available antibodies STAT.

Fig.8. Anti-IFNAR1 antibodies block the transmission of various concentrations of type I interferons derived from cells of the MPC. Presented IC50 values for the antibodies 9D4, blocking signal transmission IFN, luciferase reporter analysis using the supernatants of IFN type I, purified from three independent donors. Contains relative amounts of IFNα, IFNβ and IFNω each purified supernatant of type I interferon.

Fig.9 A, B, C. the Anti-IFNAR1 antibodies 9D4, 9D4-DM (double �of utant) and 9D4-TM (triple mutant) exhibit similar binding properties. Shown data represent an unmodified antibody 9D4 along with 2 modified antibodies 9D4-DM and 9D4-TM. The modified antibodies show similar numbers of binding of IFNAR1 with unmodified antibody.

Fig.10A. Anti-IFNAR1 antibody 9D4 binds a soluble interferon receptor alpha (soluble interferon alpha receptor - sIFNαR1). Presents equilibrium binding data, which show a dose-dependent binding 9D4 with soluble interferon alpha receptor.

Fig.10B. Determination of the Kd of the antibody 9D4 in the mononuclear cells of peripheral blood (PBMC) of a person. Shows the determination of dissociation constants of antibodies 9D4, measured by the binding to human PBMC.

Fig.11. Anti-IFNAR1 antibodies inhibit induced IFNα signal transmission in a luciferase reporter assay. Anti-IFNAR1 antibodies, including unmodified and modified antibodies that exhibit similar IC50 values for blocking of signal transmission through leukocyte IFN in the luciferase reporter system analysis.

Fig.12A. Determination of the isoelectric point of the antibody 9D4 (unmodified) and modified antibody 9D4. Shown IEF gel documenting the relative values pI for antibodies 9D4 WT (unmodified), 9D4-DM and 9D4-TM.

Fig.12B. Determination of the melting point of an antibody 9D4 (unmodified) and mod�modified antibodies 9D4. The figure presents melting curves representing the relative melting temperatures (melting temperatures Tm) for antibodies 9D4, 9D4-DM and 9D4-TM.

Fig.13. Prophylactic treatment with anti-IFNAR antibodies blocks Adv-IFNα-induced proteinuria. Mice treated with the control vector, Adv-IFNα, Adv-IFNα + pretreatment isotype control and Adv-IFNα + pre-treatment with anti-IFNAR, examined for proteinuria during 9 weeks. Mice pre-treated with anti-IFNAR antibody do not show proteinuria after administration of IFNα.

Fig.14. Prophylactic treatment with anti-IFNAR antibodies blocks the increased regulation of genes responsible for IFNα (IFIT1, IFI44, CXCL11, IFI202b, CXCL19, CXCL9), in the blood. Mice pre-treated with anti-IFNAR antibodies, do not show increased regulation of selected genes responsible for IFNα, when infected with adenovirus encoding IFN alpha, compared with mice pre-treated with a control virus, FSB or isotype control IgG. Presents relative expression of six genes known to respond to IFNα in blood samples taken from mice 3 weeks after the induction of IFNα by infection with Adv-IFNα.

Fig.15 A, B. Prophylactic treatment with anti-IFNAR antibodies blocks induced IFNα production of autoantibodies. Mice pre-treated and�t-IFNAR antibodies, do not show excessive generation of antibodies when infected with adenovirus encoding IFNα, compared with mice pre-treated with a control virus, FSB or isotype control IgG. Presents the concentration of anti-dhnk and anti-SSA/Ro in blood samples taken from mice after 6 min after the induction of IFNα by infection with Adv-IFNα.

Fig.16 A, B. Prophylactic treatment with anti-IFNAR antibodies blocks the increased regulation of cytokines in the kidneys. Mice pre-treated with anti-IFNAR antibodies, do not show increased regulation of cytokines in the kidney during infection with adenovirus encoding IFNα5, compared with mice pre-treated with a control virus, FSB or isotype control IgG. Presents the measurement of levels of IP-10 and IL-18 in samples of kidneys obtained from mice 6 weeks after the induction of IFNα infection Adv-IFNα5.

Fig.17. Prophylactic treatment with anti-IFNAR antibodies blocks induced IFN production of autoantibodies. Presented titers of antibodies to nuclear antigens (ANA) from mouse serum. Mice treated with anti-IFNAR antibodies exhibit reduced titers of ANA in the serum after administration of IFN compared to mice previously treated with a control virus, FSB or isotype control.

Fig.18. Mediated suppression of antibody development dendrocygna cells, op�seguenega plasma of SLE. Presents the results of 5 separate experiments in which IFN derived from patients with SLE, were incubated in the presence of anti-IFNAR1 antibodies 9D4, and then added to the monocytes. The presence of anti-IFNAR1 antibodies 9D4 inhibits the ability of IFN derived from patients with SLE, to induce markers CD38 and CD123 dendrocygna cells in differentiated monocytes.

Fig.19. Anti-IFNAR1 antibodies inhibit the expression of CD38, CD123 and CD86 in monocytes after stimulation of leukocyte interferon. By measuring the percent suppression of expression with a control stimulation, anti-IFNAR1 antibodies 9D4, 9D4-DM and 9D4-TM exhibit similar profiles of expression of CD38, CD123 and CD86 in differentiated monocytes.

Fig.20. The modified anti-IFNAR1 antibodies exhibit reduced binding to the Fc receptor FcγRI compared to unmodified anti-IFNAR1 antibodies. Anti-IFNAR1 antibody 9D4 (unmodified), 9D4-DM (modified) and 9D4-TM (modified) was analyzed for ability to bind to the tablet, which is connected with a receptor FcγRI, in the ELISA experiment. As a positive control for binding of Fc receptor using an unmodied unrelated antibody (control antibody).

Fig.21 A, B, C. the Modified anti-IFNAR1 antibodies exhibit reduced binding to the Fc receptor FcγRIIIA compared with unmodified anti-IFNAR1 EN�itelli. Associated with tablet modified anti-IFNAR1 antibody 9D4(A) and the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM(C) was analyzed for ability to bind free FcγRIIIA receptor in experimental ELISA format.

Fig.22 A, B, C. the Modified anti-IFNAR1 antibodies exhibit reduced binding to the Fc receptor FcγRIIIA. Free modified anti-IFNAR1 antibody 9D4(A) and the modified anti-IFNAR1 antibodies 9D4-DM(B) and 9D4-TM(C) was analyzed for ability to bind associated with the tablet FcγRIIIA receptor in experimental ELISA format.

Fig.23 A-D. Neutralization of IFN subtypes in the serum of patients with SLE. According to the reporter measurements in the analysis of anti-IFNAR1 antibody MDX-1333, 9D4-WT and 9D4-TM inhibit mediated IFN signal transmission α10 (A), leukocyte interferon (B) A2B (C), ω (G) and β (E).

Fig.24. Anti-IFNAR1 antibody neutralizes interferon type I from patients with SLE. With the help of reporter analysis showed that the anti-IFNAR1 antibody 9D4, suppresses mediated type I interferon signal transmission compared with the control, an unrelated antibody.

Fig.25 A-d. Anti-IFNAR antibodies inhibit induced IFNα population among MPC ciav. Anti-IFNAR antibodies block the increase of cells MPC installed on the expression of the epitope on the cell surface, induced ectopic adenoviral expression caused by expressie� interferon-alpha in the spleen (A), the lymph nodes (B), peripheral blood (B) and bone marrow (D).

Fig.26. Analysis of the binding of anti-IFNAR1 antibodies 9D4-WT, 9D4-DM and 9D4-TM to the Fc receptor FcγRI determined by the BIACore method. Briefly, anti-IFNAR1 antibodies immobilized and add free FcγRI to measure affinity. As follows from the figure that the modified antibodies 9D4-DM and 9D4-TM exhibit a low affinity with a free FcγRI as compared with unmodified antibody 9D4-WT.

Fig.27 A-C. Analysis of the binding of anti-IFNAR1 antibodies 9D4-WT, 9D4-DM and 9D4-TM to the Fc receptor FcγRI determined by the BIACore method. In short, the available anti-IFNAR1 antibodies passed through immobilized FcγRI to measure affinity. From the figures it follows that the modified antibodies 9D4-DM (B) and 9D4-TM (B) show a reduced affinity associated with FcγRI as compared with unmodified antibody 9D4-WT (A).

Fig.28. Anti-IFNAR antibodies inhibit IFNα-responsive gene induction in the kidneys. Briefly, in a mice model of increased manifestations of lupus, treatment with anti-IFNAR antibodies blocks the induction in the kidney of six genes (ICAM1, VCAM1, CXCL9, CXCL10 and IFIT1), mediated ectopiceski murine IFNα, compared to control mice by measuring the Taqman method.

Fig.29. Anti-IFNAR antibodies inhibit the production of anti-dndn antibodies in mice models of increased manifestations of lupus. Briefly, mice, ectopiceski expressing IFNα and processing�by anti-IFNAR antibodies, not accumulate anti-dndn antibodies to the same level as that in mice similarly infected and treated with a control IgG antibody.

Fig.30. Anti-IFNAR antibodies able to reduce proteinuria in therapeutic treatment of mice models of increased manifestations of lupus. (A) Briefly, mice with symptoms of lupus, formed under the influence of a murine IFNα, such as proteinuria. In therapeutic study, the anti-IFNAR antibody administered to mice at achievement of threshold estimates corresponding to the proteinuria. Anti-IFNAR antibodies, PBS or a control IgG administered twice a week during the course, duration of 5 weeks. Animals in the treatment group anti-IFNAR antibody is manifested reduced the severity of proteinuria during the experiment compared with the animals in groups of application of the FSB or control IgG.

Fig.31. Anti-IFNAR antibodies is able to increase the survival rate for therapeutic treatment of mice models of increased manifestations of lupus. (A) Briefly, mice having ectopiceski murine IFNα, have a reduced survival rate, component about 8 weeks after the onset of symptoms, resembling symptoms of lupus, such as the proteinuria. In therapeutic study, the anti-IFNAR antibody administered to mice after reaching the threshold of proteinuria. Anti-IFNAR antibodies, PBS or a control IgG input�t twice a week for 5 week course. After 5 weeks of antibody treatment was stopped and mortality traces for all three treatment groups. In the treatment group anti-IFNAR antibody is much lower mortality compared with the group that used only the FSB, or groups of control IgG, each fitted with 100% mortality by week 9.

Fig.32. The content view of the asymmetric unit of the crystals of the field of Fc-TM, which includes mutations L234F/L235E/P331S. Mutation R331 marked in red. One zinc ion is chelated by two spatially contiguous histidine residues. Carbohydrate residues attached at position 297, is modeled on the basis of their electron density.

Fig.33. Kinetic images show the internalization of antibodies 9D4-TM. Cells THP-1 were stained with 1 μm CFSE in the incubator at 37°C in atmosphere CO2for 10 min, followed by treatment with 1 μg/ml Alexa647-9D4-TM on ice for 1 h. After removal of the unbound reagent, the cells were incubated at 37°C for a designated time (0, 15, 30 and 60 min) and receive images taken of cells.

Fig.34. Anti-IFNAR1 antibody 9D4-TM does not show the actions of the CDC in assessing in vitro. The figure shows the results of the analysis by the CDC to determine the ability of the antibody 9D4-TM to call the CDC. From the figures it follows that 9D4-TM antibody does not show any CDC activity compared to positive�positive control antibody. Action CDC also is not detected for an unrelated control antibody R347. Briefly, cells expressing the antigen IFNAR1, or incubated with the positive control antibody 9D4-TM, or R347. After a series of washes add freshly prepared human serum. Complement-dependent cytotoxicity (CDC) was measured using the analysis of the release of lactate dehydrogenase (LDH).

Terminology

The term "interferon alpha", "IFNα", "IFNa", "IFNA" and "IFN alpha" in the present invention are used interchangeably, and they mean protein IFN alpha, encoded by a functional gene of the locus of the gene of interferon Alfa with 75% or higher sequence identity with the sequence of IFN alpha 1 (ID NP_076918 in GenBank, the protein encoded identification number NM_024013 in GenBank). Examples of subtypes of IFN alpha are IFN alpha 1, alpha 2A, alpha 2b, alpha 4, alpha 4b alpha 5, alpha 6, alpha 7, alpha 8, alpha 10, alpha 13, alpha 14, alpha 16 alpha 17 alpha 21. The term "interferon alpha", "IFNα" and "IFN alpha" refers to recombinant forms of different subtypes of IFN alpha, as well as natural products, which include protein IFN alpha, for example leukocyte IFN and lymphoblastoid IFN.

The concept of "receptor-1 interferon-alpha", "IFNAR1", "IFNAR-1 and antigen IFNAR-1" are used interchangeably; these include variants, isoforms, homologues of IFNAR-1 human�and from other types and analogues, having at least one common epitope with IFNAR-1. Thus, antibodies of the present invention may in some embodiments of the present invention cross-react with IFNAR-1 of other species (i.e. occurring not from man) or with other proteins that are structurally similar with IFNAR-1 person (for example, the homologues of IFNAR-1 human). In other embodiments of the present invention, the antibodies may be completely specific for IFNAR-1 human and not show species, or another type of cross-reactivity. The complete cDNA sequence IFNAR-1 person has an ID number NM_000629 in Genbank.

In the context of the present invention, the term "conservative modification sequence includes amino acid modifications that do not affect or modify indicators of binding of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitution, insertion, and deletion. Modifications can be introduced into an antibody of the present invention using standard methods known in this field, for example, site-directed mutagenesis and PCR-mediated mutagenesis. Conservative are amino acid substitution in which the amino acid residue substituted amino acid residue with �Hoza side chain. For example, one or more amino acids with similar polarity act as functional equivalents and lead to napravljena change in the amino acid sequence of the peptide. Of substitution, which is neutral in charge and replaces the balance on a smaller residue may also be considered "conservative substitutions" even if the residues are in different groups (for example, substitution of phenylalanine for a smaller isoleucine). Family of amino acid residues having similar side chains are known and identified in the field. Several non-limiting examples of families of amino acids shown in table.1.

Table 1.
Families of conservative amino acid substitutions.
FamilyAmino acids
NonpolarTrp, Phe, Met, Leu, Ile, Val, Ala, Pro
Uncharged polarGly, Ser, Thr, Asn, Gln, Tyr, Cys
Acidic/negatively chargedAsp, Glu
Basic/positively chargedArg, Ly, His
Beta-branchedThr, Val, Ile
Residues that influence chain orientationGly, Pro
AromaticTrp, Tyr, Phe, His,

Detailed description of the present invention

Unlike previous developments in the present invention it was found that the anti-IFNAR1 antibodies with reduced or truncated effector function is desirable for the treatment of chronic autoimmune and/or inflammatory diseases. Previously had developed antibodies directed against IFNAR1, this meant that the effector function may play a role in mediating cure or at least alleviate the condition of chronic autoimmune and/or inflammatory disease (see, for example, the publication US 20060029601 or PCT WO 06002177). According to this concept, many previous developments have been aimed at identifying anti-IFNAR1 antibodies with strong effector function will be further enhanced effector function by increasing the affinity of the antibody with Fc receptors (e.g., FcRn, FcγRIIIa, FcγRIIb) and/or protein of complement C1q. Formed the opinion that such the resulting anti-IFNAR1 antibodies with increased effector function is useful in the treatment of these bol�znanych States.

Unlike the earlier of such opinion, the present invention describes anti-IFNAR1 antibodies with reduced or truncated effector function (e.g., cytotoxicity ADCC and/or CDC). Through studies tissue cross-reactivity unexpectedly it was found that the anti-IFNAR1 antibodies with strong or increased effector function possess the predisposition to unwanted toxicity due to the prevalence of staining with anti-IFNAR1 on fabrics, not targeted. Such toxicity may arise from nonspecific activation of ADCC and/or CDC of non-compliant sites. To reduce or eliminate such undesirable toxicity was the necessity to reduce the effector function of the polypeptide comprising the Fc region.

Thus, one object of the present invention encompasses modified antibodies or other polypeptides comprising the Fc region of the antibody containing the insertion, substitution or deletion of at least one amino acid residue of the Fc region, resulting in decreased or the truncated affinity in relation to at least one Fc ligand (denoted in the present invention "modified antibodies of the present invention", "modified antibody" or "antibody of the present invention"). The Fc region interacting� with a number of ligands, including, but not limited to, Fc receptors (e.g., FcRn, FcγRIIIa, FcγRIIb), a protein of the complement C1q and other molecules, e.g., proteins A and G. These interactions are essential for various effector functions, and the subsequent events in the signal transmission, including, but not limited to, antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, in some embodiments of the present invention, the modified antibodies of the present invention have low or reduced affinity against Fc ligand that is associated with relief effector function compared to an antibody having the same amino acid sequence as the antibody of the present invention, but not including additions, substitutions or deletions of at least one amino acid residue of the Fc region (also referred to in the present invention "non-modified antibody"). In some embodiments of the present invention the antibodies of the present invention include at least one or more of the following signs: reduced or truncated effector (ADCC and/or CDC) function, reduced or reduced binding to Fc receptors, or reduced or truncated toxicity. More specifically, embodiments of the truly magnificent�meniu provide anti-IFNAR1 antibodies with reduced affinity to Fc receptors (e.g., FcRn, FcγRIIIa, FcγRIIb) and/or protein of complement C1q.

In one of the embodiments of the present invention the antibodies of the present invention comprise an Fc region comprising at least one addition, substitution or deletion of amino acid residues chosen from the provisions 234, 235 and 331, and the numbering system of the constant region is given according to the EU index, developed by Kabat and others (Publication of the National Institute of health, USA 91-3242, 1991, national technical information service, Springfield, Virginia). In some embodiments of the present invention the antibodies of the present invention comprise an Fc region containing at least one amino acid substitution selected from the group consisting of: L234F, L235E, and P331S, with the first letter and number means either the unmodified amino acid and its position, and the second letter represents a substituted amino acid at the specified position.

In another embodiment of the present invention the antibodies of the present invention further comprise an Fc region containing at least one addition, substitution or deletion of amino acid residues, which correlates with increased stability of the antibody. In one embodiment of the present invention, the addition, substitution or divide the amino acid residue located in position�AI 228 Fc region, moreover, the numbering system of the constant region is given according to the EU index, developed by Kabat and others In certain embodiments, antibodies of the present invention comprise an Fc region containing the amino acid substitution at position 228, wherein the substitution is carried out on a serine residue. In another embodiment of the present invention the antibodies of the present invention IgG4 subtype include amino acid substitution of serine at position 228 Fc region. In other embodiments of the present invention the antibodies of the present invention include the residue serine at position 228 Fc region; in such embodiments, modifications are not required. In other embodiments, antibodies of the present invention do not require modification of residue 228 Fc region or already contain a serine at the position.

In another embodiment of the present invention the antibodies of the present invention can be any of the antibodies of any subclass (e.g., but their list is not limited to, IgG, IgM, and IgE). In some embodiments of the present invention the antibodies of the present invention are members of a class of IgG antibodies. In some embodiments, the antibodies of the present invention are of the IgG1 subclass. In another embodiment, the implemented�I of the present invention the antibodies of the present invention are of the IgG1 subclass and include the following amino acid substitution: 234F, E and 331S Fc region. In other embodiments, antibodies of the present invention belong to the IgG4 subclass. In certain embodiments, the antibodies of the present invention belong to the IgG4 subclass and include the following amino acid substitution: S228P and L235E Fc region.

In some embodiments of the present invention, the modified antibodies of the present invention can be produced by combining variable domain or fragment of the Fc domain comprising one or more amino acid substitutions, as described in the present invention. In other embodiments of the present invention, the modified antibodies of the present invention can be produced by modification of the antibody containing the Fc domain, by introducing one or more substitute amino acid residues in the Fc domain.

Reduced binding to Fc ligands

For the person skilled in the art it is obvious that the antibodies of the present invention can be modified (relative to the unmodified antibody) FcγR and/or C1q binding properties (examples of binding properties include but not limited to, binding specificity, equilibrium dissociation constant (KD), degree of dissociation and Association (Koffand Konrespectively), binding�e affinity and/or avidity) and that certain changes are more or less desirable. In this area it is known that the equilibrium dissociation constant (KD) is expressed as koff/kon. The person skilled in the art can determine the kinetic parameter is most important for the application of this antibody. For example, a modification that reduces binding to one or more positive regulators (e.g., FcγRIIIA), and/or improves the binding to an inhibitory Fc receptor (e.g., FcγRIIB), can be applied to reduce the action of ADCC. Thus, the degree of binding affinity (i.e., the equilibrium constants of dissociation (KD)) can indicate if the ADCC activity of the antibody of the present invention raised or lowered. In addition, a modification that reduces binding to C1q may be applicable to reduce or eliminate the actions of the CDC.

The affinities and binding properties of the Fc region with its ligand can be determined by various methods in vitro studies (biochemical or immunological), known in this area to determine the interactions of Fc-FcγR, i.e., specific binding of the Fc region to an FcγR receptor, including, but not limited to, equilibrium methods (e.g. enzyme-linked linked immunosorbent assays (ELISA) or a radioimmunoassay (RIA)), or kinetic methods (e.g., BIACORE®) and other methods, for example, indirect methods research�hardware binding, methods of competitive inhibition, the energy transfer fluorescent resonance (fluorescence resonance energy transfer - FRET), gel electrophoresis and chromatography (e.g., gel filtration). In these and other methods can use a label to one or more of the investigated components and/or apply various methods of detection, including, but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. Detailed testing of binding affinity and kinetics, see kN.: "Fundamental Immunology", 1999, edited by W. E. Paul, 4eed., publ Lippincott-Raven, Philadelphia.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced binding affinity to one or more Fc receptors including, but not limited to, receptor FcγRI (CD64), including isoforms FcγRIA, FcγRIB and FcγRIC; FcγRII (CD32 including isoforms FcγRIIA, FcγRIIB and FcγRIIC; and FcγRIII (CD16, including isoforms FcγRIIIA and FcγRIIB), as compared with unmodified antibody. In some embodiments of the present invention the antibodies of the present invention do not include a concomitant increase in binding of the receptor FcγRIIB in comparison with non-modified (e.g., containing the Fc region of the wild type) antibody.

In one of the embodiments of the present invention the antibodies of the present invention �reavley low affinity to FcγRI as compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRI, which is at least 2-fold, or at least 3 fold, or at least 5-fold, or at least 7-fold, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60 times, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100-fold, or at least 200 times less than that of the unmodified antibody.

In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRI, which is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, by at least 30%, at least 20%, at least about 10%, or by at least 5% less than that of the unmodified antibody.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced affinity to FcγRIIIA receptor as compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention show affinity with receptor� FcγRIIIA, at least in 2 times, or at least 3 fold, or at least 5-fold, or at least 7-fold, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70 fold, or at least 80 times, or at least 90 fold, or at least 100-fold, or at least 200 times less than that of the unmodified antibody.

In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRIIIA that is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, by at least 30%, at least 20%, at least about 10%, or by at least 5% less than that of the unmodified antibody.

In this area it is known that allelic variant F158V FcγRIIIA receptor has altered binding characteristics of antibodies. In one of the embodiments of the present invention the antibodies of the present invention bind with reduced affinity to FcγRIIIA receptor (F158V) compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention exhibit crodt�about the FcγRIIIA receptor (F158V), at least in 2 times, or at least 3 fold, or at least 5-fold, or at least 7-fold, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70 fold, or at least 80 times, or at least 90 fold, or at least 100-fold, or at least 200 times less than that of the unmodified antibody. In another embodiment of the present invention the antibodies of the present invention exhibit an affinity to FcγRIIIA receptor (F158V), which is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, by at least 30%, at least 20%, at least about 10%, or by at least 5% less than that of the unmodified antibody.

In another embodiment of the present invention the antibodies of the present invention exhibit high affinity to a receptor FcγRIIB as compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRIIB that is unchanged or increased by at least 2-fold, or at least 3 fold, or at least 5 times, or minicamera 7 times, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100-fold, or at least 200-fold compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRIIB, which is increased by at least 5%, at least 10%, at least about 20%, by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to the affinity of the unmodified antibody.

In another embodiment of the present invention the antibodies of the present invention show affinity to the receptors of the FcγRI, FcγRIIIA, or FcγRIIIA (F158V), which is approximately 100 nm to about 100 μm, or from about 100 nm to about 10 μm, or from about 100 nm to about 1 μm, or from about 1 nm to about 100 μm, or from about 10 nm to about 100 μm, or from about 1 μm to about 100 μm, or from about 10 μm to about 100 μm. In some embodiments of the present invention the antibodies of the present�th invention show affinity to the receptors of the FcγRI, FcγRIIIA, or FcγRIIIA (F158V), which is greater than 1 μm, 5 μm, 10 μm, greater than 25 μm, 50 μm, 100 μm or more.

In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRIIB that is approximately 100 nm to about 100 μm, or from about 100 nm to about 10 μm, or from about 100 nm to about 1 μm, or from about 1 nm to about 100 μm, or from about 10 nm to about 100 μm, or from about 1 μm to about 100 μm, or from about 10 μm to about 100 μm. In some embodiments of the present invention, antibodies of the present invention show affinity to the receptors of the FcγRI, FcγRIIIA, or FcγRIIIA (F158V), which is less than 100 microns, less than 50 microns, less than 10 microns, less than 5 μm, less than 2.5 μm, less than 1 μm, 100 nm or less, 10 nm or less.

In another embodiment of the present invention the antibodies of the present invention show affinity with the receptor FcγRIIB that is approximately 100 nm to about 100 μm, or from about 100 nm to about 10 μm, or from about 100 nm to about 1 μm, or from about 1 nm to about 100 μm, or from about 10 nm to about 100 μm, or from about 1 μm to about 100 μm, or from about 10 μm to about 100 μm. In some embodiments of the present invention, the antibodies according to�present invention show affinity to the receptors of the FcγRI, FcγRIIIA, or FcγRIIIA (F158V), which is less than 100 microns, less than 50 microns, less than 10 microns, less than 5 μm, less than 2.5 μm, less than 1 μm, 100 nm or less, 10 nm or less.

Reduced ADCC activity

In this area it is known that antibodies are able to direct the attack and destruction of the target antigen for a variety of processes, collectively known as the "effector functions of antibodies. One process, called "antibody-dependent cretaceouspaleogene cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) found on some cell cytotoxicity (e.g., natural cells killer cells (NK), neutrophils and macrophages), allowing these effector cells cytotoxicity specifically to contact with bearing the antigen on target cells and then destroy the target cell with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of target cells, "arming" of the cell cytotoxicity and is required for such destruction. Lysis of target cells extracellular requires direct contact between cells and does not involve complement. Another process that is covered by the concept of effector function is complement-dependent cytotoxicity (denoted in the present invention "CDC"), which relative�tsya the biochemical event of destruction of target cells, mediated by the complement system. The complement system is a complex system of proteins found in normal blood plasma that combines with antibodies to destroy pathogenic bacteria and other foreign cells.

Can be examined the ability of any particular antibody to mediate lysis of target cells through ADCC. To evaluate the effect studied ADCC antibody was added to target cells in combination with immune effector cells that can be activated by complexes of antigen with the antibody, leading to cytolysis of target cells. Cytolysis is usually discovered on the release of the label (e.g., radioactive substances, fluorescent dyes or natural intracellular proteins from lysed cells. Useful effector cells include mononuclear cells of peripheral blood (PBMC) cells and natural killer cells (Natural Killer - NK). Specific examples of studies in vitro ADCC described Wisecarver, etc., 79, 1985, cc.277-282; Bruggemann et, J Exp Med 166, 1987, cc.1351-1361; Wilkinson et, J Immunol Methods 258, 2001, cc.183 to 191; Patel, etc., J Immunol Methods 184, 1995, cc.29-38. In another embodiment, or additionally, ADCC activity of the investigated antibodies can be assessed in vivo, e.g., in animal models, for example, as described by dynes and others, PNAS USA 95, 1998, cc.652-656.

Assume that the antibodies of the present invention on�isany using functional in vitro studies to determine the one or more effector cell functions, mediated FcγR. In some embodiments of the present invention the antibodies of the present invention have similar binding properties and effector cell functions in in vivo models (e.g., as described in the present invention), the same as established in studies in vitro. However, the present invention does not exclude the antibodies of the present invention that do not exhibit the desired phenotype in studies based on in vitro, but actually exhibit the desired phenotype in vivo.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced ADCC activity compared to the unmodified antibody. In another embodiment of the present invention the antibodies of the present invention showing the ADCC activity that is at least 2-fold, or at least 3 fold, or at least 5 times or at least 10 fold or at least 50 times or at least 100 times less than the action of the unmodified antibody, In still another embodiment, one implementation of the present invention the antibodies of the present invention showing the ADCC activity that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50% or at least 60%, or by at least 70%, or at least 80% or at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to the unmodified antibody. In some embodiments of the present invention the antibodies of the present invention do not exhibit detectable actions ADCC. In some specific embodiments, the reduction and/or truncation of the action of ADCC may be due to a lowered affinity antibodies of the present invention that they have shown for ligands and/or receptors Fc.

Reduced CDC action

The metabolic pathway of complement activation is initiated by binding of the first component of the complement system (C1q) to a molecule, e.g., antibody, combined with a cognate antigen. To assess complement activation can be performed in a CDC study, for example, described Gazzano-Santoro et, J. Immunol. Methods, 202, 1996, p. 163.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced affinity to C1q relative to the unmodified antibody In another embodiment of the present invention the antibodies of the present invention exhibit an affinity to C1q receptor that is at least 2-fold, or at least 3 fold, or by minicamera 5 times or at least 7-fold, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100-fold, or at least 200 times less than the affinity of the unmodified antibody.

In another embodiment of the present invention the antibodies of the present invention exhibit an affinity to C1q, which is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, by at least 30%, at least 20%, at least about 10%, or by at least 5% less than the affinity of the unmodified antibody.

In another embodiment of the present invention the antibodies of the present invention exhibit an affinity to C1q, which is approximately 100 nm to about 100 μm, or from about 100 nm to about 10 μm, or from about 100 nm to about 1 μm, or from about 1 nm to about 100 μm, or from about 10 nm to about 100 μm, or from about 1 μm to about 100 μm, or from about 10 μm to about 100 μm. In some embodiments of the present invention, antibodies of the present invention exhibit an affinity to C1q, which� more than 1 μm, more than 5 microns, greater than 10 μm, greater than 25 μm, greater than 50 microns, or greater than 100 microns.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced activity CDC compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention showing the action of the CDC, at least 2 times, or at least 3 fold, or at least 5 times or at least 10 fold, or at least 50-fold, or at least 100 fold less than the activity of unmodified antibody. In still another embodiment, one implementation of the present invention the antibodies of the present invention showing the action of the CDC, which is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50% or at least 60% or at least 70%, or by at least 80% or at least 90%, or by at least 100%, or at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to the action of the unmodified antibody. In some embodiments of the present invention the antibodies of the present invention do not exhibit detectable actions CDC. In some embodiments, the OS�enjoyment of present invention, the reduction and/or truncation of the action of CDC can be attributed to a reduced affinity, which the antibodies of the present invention have shown for Fc ligands and/or receptors.

The reduced toxicity associated with the antibody

In this area it is known that biological therapy may have side toxicity associated with the complex nature guided by the immune system to recognize and attack unwanted cells and/or target. If recognition and/or targeting for attack no, but treatment is required, the consequence can be a side toxicity. For example, antibody staining of tissues which were not targeted, can be an indicator of potential toxicity.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced staining of non-target tissues compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention exhibit reduced staining of non-targeted tissue that is at least 2-fold, or at least 3 fold, or at least 5-fold, or at least 7-fold, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60 times, or at least 70 fold, or at least 80 fold, or at least 90 times, or u� least 100 times, or at least 200 fold less than the staining of the unmodified antibody. In another embodiment of the present invention the antibodies of the present invention exhibit reduced staining of non-targeted tissues, which is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50% or at least 60% or at least 70%, or by at least 80% or at least 90%, or by at least 100%, or at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to the unmodified antibody.

In one of the embodiments of the present invention the antibodies of the present invention exhibit reduced associated with the antibody toxicity compared with unmodified antibody. In another embodiment of the present invention the antibodies of the present invention exhibit toxicity, which is at least 2-fold, or at least 3 fold, or at least 5-fold, or at least 7-fold, or at least 10 fold, or at least 20-fold, or at least 30 fold, or at least 40-fold, or at least 50-fold, or at least 60 times, or at least 70 fold, or at least 80 fold, or at Merav 90 times or at least 100-fold, or at least 200 fold less than the toxicity of unmodified antibody. In another embodiment of the present invention the antibodies of the present invention exhibit toxicity that is reduced by at least 10%, or by at least 20%, or by at least 30%, or by at least 40%, or by at least 50% or at least 60% or at least 70%, or by at least 80% or at least 90%, or by at least 100%, or at least 200%, or by at least 300%, or by at least 400%, or by at least 500% compared to the toxicity of unmodified antibody.

Internalization of antibodies

The antibodies of the present invention can bind to the antigens on the surface of cells that can internalize, also bringing the antibody into the cell. Getting inside the cell, the antibodies can be released into the cytoplasm, targeted to a specific compartment or returned to the surface of the cell. In some embodiments of the present invention the antibodies of the present invention bind to the antigens on the surface of cells that internalizers. In other embodiments of the present invention the antibodies of the present invention can be directed to specific organelles or the Communist�cops cells. In other embodiments of the present invention the antibodies of the present invention can be returned to the surface of the cell or to the periphery after internalization. In one embodiment of the present invention, the antibody of the present invention is specific in respect of IFNAR1.

Internalization of antibodies can be measured adopted in this area by such methods as shown in example 34. In some embodiments of the present invention, the degree of internalization is represented as a percentage of the total number of antibodies associated with the cells. In other embodiments of the present invention, the degree of internalization of the antibody is represented by comparison with the nonspecific control antibody. In other embodiments of the present invention, the degree of internalization of the antibody present by comparing the antibody that binds to the antigen on the cell surface, the antibody that does not internalizacao. In some other embodiments of the present invention, the degree of internalization of the antibody correlates with the destruction of antibodies. In other embodiments, implementation of the present invention, the degree of internalization of the antibodies presented in the form of the ratio of staining of the cytoplasm to stain the cell surface.

In one� of embodiments of the present invention the antibodies of the present invention in the case of binding internalizing in cells moreover, internalization is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, or at least about 170% more compared to a nonspecific control antibody.

In another embodiment of the present invention the antibodies of the present invention in the case of binding internalizing in cells, and internalization is on 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170% more than a nonspecific control antibody.

In another embodiment of the present invention the antibodies of the present invention in the case of binding internalizing in cells, and internalization on 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170% more than control antibodies, according to the definition of internalization method with the use of secondary antibodies.

Three-dimensional structure region�STI Fc man

The present invention also provides crystalline forms of the Fc region of human IgG, the Fc region of a person, referred to as Fc-TM, consists of amino acid substitution L234F, L235E, and P331S, and the numbering system is given according to the EU index, developed by Kabat, and exhibits reduced or truncated effector function (ADCC and/or CDC), low or reduced binding to Fc receptors and/or reduced or truncated toxicity. In some embodiments of the present invention, the crystals differ in the orthorhombic space group S1with a unit cell a=50,18, b=147,30 and C=75,47. In some embodiments, the implementation of the present invention, the diffraction crystals have the quality to determine the three-dimensional structure of x-ray diffraction of the crystalline polypeptide (polypeptide) high resolution, preferably to a resolution of about more than 3 Å, typically in the range of from about 2 Å to about 3 Å.

The present invention also provides high-resolution three-dimensional structures and atomic structural coordinates of the crystals of Fc-TM. Specific methods used to obtain the crystals and structural coordinates shown below in the examples.

Atomic coordinates of the crystal structure of Fc-TM, obtained from the shape S1crystal with a resolution of 2.3 Å, listed� in table.6. All balances with provisions to 445 236 can be mounted on the electron density and the electron density observed for residues of the hinge region in front of the position 236, including mutations L234F and L235E, the electron Density at position 331 corresponds to serina.

The overall three-dimensional structure of Fc-TM is very similar to previously published structures elegantnyh areas Fc (Deisenhofer, Biochemistry, 20, 1981, cc.2361-2370; Krapp et, J. Mol. Biol. 325, 2003, cc.979-989; Matsumiya, etc., J. Mol. Biol. 368, 2007, cc.767-779; Oganesyan, etc., Molecular Immunology, December 11, 2007, in press). When the individual assessment of Fc-TM CH2 and CH3 domains show a high degree of structural conservatism and rigidity when compared to other elegantnymi nematandani structures of the human Fc.

Can be used information about the structure in a variety of computational or computer-based methods for screening, designing or identifying anti-IFNAR antibodies with altered biological properties. For example, the crystals and structural coordinates received from them can be used for screening, designing or identifying amino acid additions, substitutions or deletions in the Fc region that result in decreased or destructive binding to Fc receptors, decreased or destructive effector (ADCC and/or CDC) function, or lower or destructive toxicity.

�if the antibody was designed and selected using the aforementioned method, its effector function, binding to Fc receptors, or toxicity can be tested and optimized by any method known in this field. Examples of methods described in sections 5.1 to 5.4 above.

The present invention also relates to anti-IFNAR1 antibodies that are designed or selected using information about the structure of Fc-TM and which exhibit the desired biological activity. In some embodiments of the present invention such antibodies comprise an Fc region with mutations L234F, L235E and P331S. In some embodiments of the present invention such antibodies comprise an Fc region with one or more additions, substitutions or divisions amino acid residues (residues) that are not amino acid residues 234, 235 and 331.

Anti-IFNAR1 antibodies

In one of the embodiments of the present invention the antibodies of the present invention is specific (i.e., specifically bind) in respect of IFNAR1. Such antibodies can also be labeled "anti - IFNAR1 antibodies of the present invention". In another embodiment of the present invention the antibodies of the present invention is specific against human IFNAR1. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention can cross-react with IFNAR1 each�x species (but not person) or with other proteins, are structurally related to human IFNAR1 (for example, the homologues of human IFNAR1). In other embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention may be specific only in relation to human IFNAR1 and not exhibit species or other types of cross-reaktionsprodukt.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced binding affinity to Fc ligands and have at least one of the following properties: a reduced or destructive effector function (ADCC and/or CDC), reduced or destructive binding to Fc ligands, or reduced or destructive toxicity compared with unmodified antibody.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include addition, substitution or deletion of at least one amino acid residue from the group consisting of: L234F, L235E and P331S. In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include amino acid substitution: L234F, L235E and P331S in the field of Fc. In another embodiment of the present invention, the anti-IFNAR1 antibody of the present invention is an antibody of IgG isotype.

In another embodiment, the implemented�I of the present invention, the anti-IFNAR1 antibodies of the present invention belong to the IgG4 subclass. In yet another variant implementation of the present invention, the anti-IFNAR1 IgG4 antibodies of the present invention include amino acid substitution L235E in the field of Fc. In another embodiment of the present invention, the anti-IFNAR1 IgG4 antibodies of the present invention also include amino acid substitution, which correlates with increased stability. In one embodiment of the present invention, the anti-IFNAR1 IgG4 antibodies of the present invention also include amino acid substitution S228P in the field Fc.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced or destructive binding affinity to Fc receptors (e.g. FcγRI (CD64), but this receptor the list is not limited, including isoforms FcγRIA, FcγRIB and Fc-yRIC; FcγRII (CD32), including isoforms FcγRIIA, FcγRIIB, and FcγRIIC; and FcγRIII (CD16), including isoforms FcγRIIIA and FcγRIIB), as compared with unmodified antibody. In some embodiments of the present invention anti-IFNAR 1 antibodies of the present invention exhibit reduced affinity to FcγRI as compared with unmodified antibody. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced affinity to FcγRIIIA receptor relative to remodification�about antibodies. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention bind with low affinity with allele F158V FcγRIIIA relative to the unmodified antibody.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced or destructive binding affinity to C1q as compared with unmodified antibody. In one embodiment of the present invention, the anti-IFNAR 1 antibodies of the present invention exhibit reduced affinity to FcγRI relative to the unmodified antibody.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced or destructive effector function. In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced or destroyed by the action of ADCC and/or CDC. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit reduced or destructive toxicity.

The sequence of the anti-IFNAR1 antibodies

In one embodiment of the present invention, the amino acid sequence of variable regions of heavy chains and/or variable regions of light �of EPA anti-IFNAR1 antibodies of the present invention is provided in the present invention and shown in Fig.1A, 2A, 3A, 4A and Fig.1B, 2B, 3B, 4B, respectively. In yet another variant implementation of the present invention, the polynucleotide sequence that encodes the variable region of the heavy chain and variable region light chain of the anti-IFNAR1 antibodies of the present invention, provided in the present invention and shown in Fig.1A, 2A, 3A, 4A and Fig.1B, 2B, 3B, 4B, respectively.

In another embodiment of the present invention with selected sequences of the anti-IFNAR1 antibodies of the present invention can be found in the patent US 5919453, US patent applications 10/831459, 10/182058, 11/157494 and 11/521102, the essence of which is included in the present invention in the form of links. In another embodiment of the present invention the sequence of the anti-IFNAR1 antibodies of the present invention do not include the sequences described in the patent US 5919453, US patent applications 10/831459, 10/182058, 11/157494 and 11/521102.

In other embodiments of the present invention the antibodies of the present invention described in patent applications US 60/842925, 60/866917, 60/911397, 60/915309, 11/852106 and PCT US2007/07791, the essence of which is included in the present invention in the form of links.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention also include antibodies that include amino acid sequentially�th variable heavy chain and/or variable light chain, which is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of variable heavy chain and/or light chain of antibodies 3F11, E, 4G5, and 9D4 (see sequence in Fig.1-4).

Note that the number of residues of the regions, complementarity determining (CDR), referred to in the present description, are given by the classification of Kabat and others (Publication of the National Institute of health, USA 91-3242, 1991, national technical information service, Springfield, Vancouver). Specifically, residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the variable domain light chain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the variable domain of the heavy chain. It is noted that region CDR significantly vary from antibody to antibody (and by definition do not show homology with the consensus sequences in Kabat). The maximum alignment of residues wireframe plot often requires insertion "GS spacer" residues in the numbering system used for the field Fv. Note that referred to in the present invention the field of the CDR presented above in the numbering according to Kabat, etc. in addition, the identity of certain individual adhering to the shaft�in any given room site for Kabat can vary from chain antibody chain antibodies due to cross-species or allelic divergence.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include at least one region of a VH CDR having the amino acid sequence of any of the CDR sequences listed in table.2. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include at least one region of a VL CDR having the amino acid sequence of any of the sequences of the VL CDRs listed in table.2. In other embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention include one or more regions of the VH CDRs and one or more regions of a VL CDR listed in table.2. In still some other embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention include any combination of fields VH CDR and VL CDR listed in table.2. In yet another variant implementation of the present invention, the anti-IFNAR1 antibodies of the present invention can include at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least the 6 CDRs selected from the table.2. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention may include a VH domain and/or VL domain, each of �which includes 1, 2 or 3 CDR. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention may include a VH region, optionally containing 1, 2 or 3 region CDR (CDRH#) heavy chains listed in table.2. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention can include a VL region, optionally containing 1, 2 or 3 region light chain CDR (cdrl stock#) listed in table.2.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include the CDR of the antibody 3F11 (see, e.g., PL.2). In yet another variant implementation of the present invention, the anti-IFNAR1 antibodies of the present invention include the CDR of the antibody 4G5 (see, for example table.2). In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include the antibody CDR E (see, for example table.2). In yet another variant implementation of the present invention, the anti-IFNAR1 antibodies of the present invention include the CDR of the antibody 9D4 (see, e.g., PL.2).

14
Table 2.
CDR sequences of the anti-IFNAR1 antibodies
Antibody CDRSequenceSeq ID No:
3F11CDRL1RASQGIYSVLA1
3F11CDRL2DASRLES2
3F11CDRL3QQFNSYIT3
3F11CDRH1GYFWS4
3F11CDRH2EIDHSGKTNYNPSLKS5
3F11CDRH3ESKYYFGLDV6
4G5CDRL1RATQDISIALV11
4G5CDRL2DASGLGS12
4G5CDRL3QQFNSYPYT13
4G5CDRH1NYYWS
4G5CDRH2EIILSGSTNYNPSLKS15
4G5CDRH3ESKWGYYFDS16
ECDRL1RASQSVSSSFFA21
ECDRL2GASSRAT22
ECDRL3QQYYDSSAIT23
ECDRH1NYWIA24

AntibodyCDRSequenceSeq ID No:
ECDRH2IIYPGDSDIRYSPSFQG25
ECDRH3HDIEGFDY26
9D4CDRL1 RASQSVSSSFFA31
9D4CDRL2GASSRAT32
9D4CDRL3QQYDSSAIT33
9D4CDRH1NYWIA34
9D4CDRH2IIYPGDSDIRYSPSFQG35
9D4CDRH3HDIEGFDY36

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include the amino acid sequence of the variable heavy chain and/or variable light chain that include at least 1 at least 2 at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 amino acid substitutions, additions or divisions compared with the variable heavy chain and/or variable light chain, represented in Fig.1, 2, 3 or 4. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention �fied one or more CDR with at least 1, at least 2, at least 3, at least 4, at least 5 or at least 10 amino acid substitutions, divisions or additions in one or more CDR regions listed in table.2.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include antibody encoded by the polynucleotide sequence that's hybrid with the nucleotide sequence shown in Fig.1, 2, 3, or 4 under stringent conditions. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention comprise one or more CDR regions encoded by a nucleotide sequence that hybrid under stringent conditions with the nucleotide sequence of one or more CDR regions listed in Fig.1, 2, 3 or 4. The conditions are stringent hybridization include, but are not limited to, hybridization with related filter DNA in 6x sodium chloride/sodium the citrate (SSC) at about 45°C followed by one or more washes in 0.2×SSC/0,1% SDS at about 50-65°C, under conditions of high stringency, for example, hybridization with related filter DNA in 6×SSC at about 45°C followed by one or more washes in 0.1×SSC/0.2% of SDS at about 60°C, or any other hard conditions of hybridization, �known to specialists in this field (see, for example, kN.: "Current Protocols in Molecular Biology", 1989, ed. by Ausubel P. M., etc., vol. 1, ed. Green Publishing Associates, Inc. and John Wiley and Sons, Inc., New York, cc.6.3.1-6.3.6, 2.10.3). In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention include, but are not limited to, antibodies encoded by a polynucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the polynucleotide sequences encoding the antibodies 3F11, E, 4G5 or 9D4 (see, Fig.1-4).

Binding affinity of anti-IFNAR1 antibodies

In some embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit a high binding affinity to IFNAR1. In some embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention showing the degree of Association (kon) constituting at least 105M-1h-1at least 5×105M-h-1at least 106M-1h-1at least 5×106M-1h-1at least 107M-1h-1at least 5×107M-1h-1or at least 108M-1 h-1. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit a value of konconstituting at least 2×105M-1h-1at least 5×105M-1h-1at least 106M-1h-1at least 5×106M-1h-1at least 107M-1h-1at least 5×107M-1h-1or at least 108M-1h-1.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention showing the degree of dissociation (koffin less than 10-1h-1less than 5×10-1h-1less than 10-2h-1less than 5×10-2h-1less than 10-3h-1less than 5×10-3h-1less than 10-4h-1less than 5×10-4h-1less than 10-5h-1less than 5×10-5h-1less than 10-6h-1less than 5×10-6h-1less than 10-7h-1less than 5×10-7h-1less than 10-8h-1less than 5×10-8h-1less than 10-9h-1less than 5×10-9h-1or less than 10-10-1h-1. In another embodiment, the implemented�I of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit a k offless than 5×10-4h-1less than 10-5h-1less than 5×10-5h-1less than 10-6h-1less than 5×10-6h-1less than 10-7h-1less than 5×10-7h-1less than 10-8h-1less than 5×10-8h-1less than 10-9h-1less than 5×10-9h-1or less than 10-10h-1.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit an affinity constant or Ka(kon/koff) constituting at least 102M-1at least 5×102M-1at least 103M-1at least 5×103M-1at least 104M-1at least 5×104M-1at least 105M-1at least 5×105M-1at least 106M-1at least 5×106M-1at least 107M-1at least 5×107M-1at least 108M-1at least 5×108M-1at least 109M-1at least 5×109M-1at least 1010M-1at least 5×101M-1at least 1011M-1at least 5×1011M-1 at least 1012M-1at least 5×1012M, at least 1013M-1at least 5×1013M-1at least 1014M-1at least 5×1014M-1at least 1015M-1or at least 5×1015M-1.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit a dissociation constant or KD(koff/kon) constituting less than 10-2M, less than 5×10-2M, less than 10-3M, less than 5×10-3M, less than 10-4M, less than 5×10-4M, less than 10-5M, less than 5×10-5M, less than 10-6M, less than 5×10-6M, less than 10-7M, less than 5×10-7M, less than 10-8M, less than 5×10-8M, less than 10-9M, less than 5×10-9M, less than 10-10M, less than 5×10-10M, less than 10-11M, less than 5×10-11M, less than 10-12M, less than 5×10-12M, less than 10-13M, less than 5×10-13M, less than 10-14M, less than 5×10-14M, less than 10-15M or less than 5×10-15M.

Specificity subtypes of interferon alpha

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effect of one or more interferons (IFN) type I, VK�including, but not limited to, IFNα, IFNβ and IFNω. Binding of IFNα subtypes can be determined using conventional methods of competition, for example, described in the book: "Antibodies: A Laboratory Manual", CSHL. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effects of interferons, including but not limited to, IFNα, IFNβ and IFNω. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effect of one or more IFNα subtypes, including, but not limited to, IFNα subtypes 1, 2A, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of IFNα subtypes. In this context, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block the binding and/or neutralizing the biological activity of IFNα subtypes: IFNα 1, 2A, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21. In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention do not exhibit the ability to block swazilan�e IFNAR1 and/or neutralize the biological effect of one or more subtypes of IFNα, including, but not limited to, IFNα subtypes 1, 2A, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21. In some embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effect of all IFNα subtypes, except IFNα21.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize a biological activity of at least 1 at least 2 at least 3 at least 4 at least 5 at least 6 at least 7 at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 of the following IFNα subtypes: 1, 2A, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21. In other embodiments, the anti-IFNAR1 antibodies of the present invention do not exhibit the ability to block binding to IFNAR1 and/or neutralize a biological activity of at least 1 at least 2 at least 3 at least 4 at least 5 at least 6 at least 7 at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 of the following IFNα subtypes: 1, 2A, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17 and 21.

In other embodiments, us�Mr sage of the invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effect of unnatural interferons, similar to type I interferons. Such unnatural type I interferons, or hybrid interferons, like type I interferons, represent a molecule that has been modified in comparison with their natural structures methods of recombination or synthesis. Hybrid interferons described in US 7232563 represent molecular substitution of various segments of the structure of natural interferon to create molecules with enhanced strength and/or reduced toxicity.

In other embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effects of mutant type I interferons. Mutant type I interferons are described in US 6299870 and 6300474, the essence of which is included in the present invention in the form of links.

In other embodiments of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological effects of interferons like IFN type I originating from other animal species. Such interferons, similar to the type I interferons isolated from chickens, cats, mice, rats, rabbits, goats, horses or animals of other species. In some embodiments, the present invention interfer�HN type I isolated from human cells, obtained from chickens, cats, mice, rats, rabbits, goats, horses, or animals of other species. In other embodiments, implementation of the present invention, the interferon type I person cause different glycosylation variants, if they are derived from chickens, cats, mice, rats, rabbits, goats, horses, or animals of other species. Additional discussion of interferons from other animal species can be found in the publication of the world organization for the protection of intellectual property WO06099451A3 included in the present invention by reference.

For problems solved in the present invention, the ability of anti-IFNAR1 antibodies of the present invention to neutralize the effect of IFNα can be monitored, for example, by the method of the study the activation of the receptor kinase (Kinase Receptor Activation - KIRA), described in WO 95/14930, by measuring the ability of the antibody candidate to reduce tyrosine phosphorylation (resulting from the binding of ligand) complex of IFNAR1/R2 receptor.

In another embodiment, or optional, for problems solved in the present invention, the ability of anti-IFNAR1 antibodies of the present invention to neutralize competition cell response via IFNα can be tested by monitoring the neutralization of IFNα antiviral activity as described by Kawade, J. Interferon Res. 1, 1980, 61, p. 70, or Kawade, Watanabe, J. InterferonRes. 4, 1984, cc.571 584, or Yousefi, etc., Am. J. Clin. Pathol. 83, 1985, cc.735-740, or by testing the ability of anti-IFNAR1 antibodies of the present invention to neutralize the ability of IFNα to activate the binding of the signaling molecule, interferon-stimulated factor 3 (interferon-stimulated factor 3 - ISGF3), to an oligonucleotide derived from the interferon-stimulated response element (interferon-stimulated response element - ISRE), in the analysis of electrophoretic mobility shift description Kurabayashi, etc., Mol. Cell Biol., 15, 1995, pp. 6386.

In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention exhibit the ability to inhibit at least one IFNα-mediated function of the receptor IFNAR1. In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention inhibit the action of the receptor IFNAR1 in response to IFNα or subtypes of at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention inhibit the action of the receptor IFNAR1 in response to IFNα or subtypes according to measurement by the method of K. IRA described in�more at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention inhibit the action of the receptor IFNAR1 in response to IFNα or subtypes according to the measure of binding of the signaling molecule, interferon-stimulated factor 3 (ISGF3), to an oligonucleotide derived from the interferon-stimulated response element (ISRE), in the analysis of electrophoretic mobility shift description Kurabayashi, etc., Mol. Cell Biol., 15, 1995, pp. 6386, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention inhibit the action of the receptor IFNAR1 in response to IFNα or subtypes according to measurements known in this field method, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about n� 85%, at least about 90%, at least about 95%, or at least about 99%.

In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention show the ability to neutralize IFNα antiviral properties, or its subtypes. In one embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention neutralize at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the antiviral action of IFNα or subtypes that were installed in antiviral research Kawade (1980) or Yousefi (1985). In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention do not neutralize IFNα antiviral properties, or its subtypes.

To solve the problem put forward in the present invention, the ability of anti-IFNAR1 antibodies of the present invention to block binding of IFNα or subtypes with IFNAR1 can be measured routine study competition, for example, described in the book: "Antibodies: A Laboratory Manual", 1988, ed. Cold Spring Harbor Laboratory, ed Harlow and David Lane. In one embodiment of the present invention, the anti-IFNAR1 the antibodies�and the present invention exhibit the ability to block or inhibit the binding of the following IFNα subtypes: 1, 2, 4, 5, 8, 10 and 21 with IFNAR1. In another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention is able to block or inhibit the binding of: at least 1 at least 2 at least 3, at least 4, at least 5, at least 6, or at least 7 of the following subtypes of IFNα: 1, 2, 4, 5, 8, 10 and 21 with IFNAR1.

The antibodies of the present invention can affect IFNAR for the regulation of IFN-1-responsible genes. IFN-1-responsible genes have been identified in the following patent applications: US60/873008, 60/907762, 60/924 584, 60/960187, 60/966 176 and PCT/US2007/02494, the essence of each of which are incorporated into this description by reference.

Antibodies

The antibodies of the present invention may include monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies, kallidinogenase antibodies, chimeric antibodies, single-chain Fvs (single-chain scFv), disulfide-linked (disulfide-linked Fvs - sdFv), anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above compounds. In particular to antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain a binding site of the antigen, and these fragments can gibridizatsiya or not to gibridizatsiya with another domain immunoglobulin�and, including, but not limited to, an Fc region or a fragment of it. As was mentioned above, the term "antibody" and "antibodies" especially include the modified antibodies described in the present invention. The immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibodies of the present invention can relate to any isotype. In one of the embodiments of the present invention the antibodies of the present invention are IgG1, IgG2, IgG3 or the IgG4 isotype. The antibodies of the present invention can be a full length antibody comprising the variable and constant region or antigen-binding fragments, e.g., single-chain antibody.

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") in the context of the present invention relates to one or more fragments of an antibody that retain the ability to specifically contact the antigen (e.g., IFNAR1). It was shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments, denoted by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of domains of the VLVH, CL/sub> and CH1(ii) a fragment F(ab')2, a bivalent fragment comprising two Fab fragments linked by disulfide bridge at the hinge region; (iii) a Fd fragment consisting of domains of the VHand CH1; (iv) a Fv fragment consisting of domains of the VLand VHone arm of an antibody; (v) a dAb fragment (Ward et, Nature 341, 1989, cc.544-546, consisting of domain VH; and (vi) the selected area, determining complementariness (CDR). Furthermore, although the two domains of the Fv fragment, VLand VHare encoded by two different genes, they can be connected using recombination methods, by a synthetic linker that enables them to be a single protein chain in which the region VLand VHconnected to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et Science 242, 1988, cc.423-426; and Huston et Proc. Natl. Acad. Sci. USA 85, 1988, cc.5879-5883). Such single-chain antibodies also include the concept of "antigen-binding fragment" of an antibody. Such antibody fragments get using traditional methods known to experts in this field, and the fragments subjected to screening in the same manner as intact antibodies.

In other embodiments, the present invention provides hybrid proteins (called hybrid proteins of the present invention") including a modified about�part Fc with reduced or altered affinity to an Fc ligand, responsible for facilitating effector function compared to an Fc region having the same amino acid sequence, and a fusion protein of the present invention, but having the addition, substitution or deletion of at least one amino acid residue in the Fc region.

In other embodiments of the present invention, the hybrid proteins of the present invention can include a peptide, polypeptide, carcass protein, scFv, dsFv, bivalent antibodies, tandem antibody (Tandab) or a mimetic antibody, hybridized with a modified Fc region. In some embodiments of the present invention, the hybrid proteins of the present invention include a linker region connecting peptide, polypeptide, frame protein (scaffold), scFv, dsFv, bivalent antibodies, Tandab, or a mimetic antibodies with modified Fc region. The use of natural as well as artificial peptide linkers to connect polypeptides into new associated hybrid polypeptides known from the literature (Hallewell et J. YOU. Chem. 264, 1989, cc.5260-5268; Alfthan, etc. Protein Eng. 8, 1995, cc.725-731; Robinson & Sauer, Biochemistry 35, 1996, cc.109-116; Khandekar et J. Biol. Chem. 272, 1997, cc.32190-32197; Fares, etc. Endocrinology 139, 1998, cc.2459-2464; Smallshaw et Protein Eng. 12, 1999, cc.623-630; US 5856456).

In one embodiment of the present invention, the hybrid proteins of the present invention include the Fc region, in�lucuuu at least one addition, substitution or deletion of amino acid residues selected from the group consisting of: 234, 235 and 331, and the numbering system of the constant region is given according to the EU index, developed by Kabat and others (Publication of the National Institute of health, USA 91-3242, 1991, national technical information service, Springfield, Virginia). In one embodiment of the present invention, the hybrid proteins of the present invention comprise an Fc region comprising at least one amino acid residue selected from the group consisting of: L234F, L235E and P331S.

In another embodiment of the present invention, the hybrid proteins of the present invention also include the Fc region comprising at least one addition, substitution or deletion of amino acid residues, which correlates with increased stability of the hybrid protein. In one embodiment of the present invention, the addition, substitution or deletion of amino acid residues contained in the position 228 Fc region, wherein the numbering system of the constant region corresponds to the EU index, developed by Kabat and Kabat and others (see above). In some embodiments of the present invention, the hybrid proteins of the present invention comprise an Fc region containing the amino acid substitution at position 228, wherein the substitution is a serine residue.

In some embodiments of the present invention the antibody or hybrid proteins of the present invention comprise one or more engineered glycoforms, i.e. carbohydrate compositions, which are covalently attached to a molecule comprising an Fc region. Designed glycoform can be used for different purposes, including but not limited to, loss of effector functions. Designed glycoform can be obtained by any method known to specialists in this field, for example by using engineered or variant expression strains, joint expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTIll), expression of a molecule comprising an Fc region in various organisms and cell lines from various organisms, or by modification of carbohydrate (carbs) after the expression of molecules including the Fc region. Methods of obtaining design glycoforms are known in this field and include, but are not limited to, the methods described in Umana et al, Nat. Biotechnol 17, 1999, cc.176-180; Davies et, Biotechnol Bioeng 74, 2001, cc.288-294; Shields et al, J Biol Chem 277, 2002, cc.26733-26740; Shinkawa et, J Biol Chem 278, 2003, cc.3466-3473) US 6602684; US 10/277370; US 10/113929; PCT WO 00/A; PCT WO 01/A; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (firm Biowa, Inc. Princeton, NJ); GlycoMAb™ technology design �of glikozilirovanie (company GLYCART biotechnology AG, Zurich, Switzerland); the essence of each of these activities is included in the present invention by reference. See, for example, the publication WO 00061739; EA; US 20030115614; Okazaki and others, JMB, 336, 2004, cc.1239-49, each of which is included in the present invention by reference.

Conjugates of antibodies

The present invention encompasses the use of antibodies or fragments thereof, conjugated or hybridized with one or more parts of molecules, including but not limited to, peptides, polypeptide, proteins, hybrid proteins, nucleic acid molecules, low molecular weight compounds, agents-mimetics, synthetic drugs, inorganic molecules and organic molecules.

The present invention encompasses the use of antibodies or fragments thereof, recombinante hybridized or chemically conjugated (including both covalent and non-covalent conjugates) with a heterologous protein or polypeptide (or fragment of a polypeptide comprising at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to produce hybrid proteins. Hybrids need not be straight, they may have a connection via a linker sequence. For example, antibodies can inform�seek to target heterologous polypeptides to specific cell types, or in vitro, or in vivo, or using conjugarea antibodies with antibodies that are specific to certain receptors on the cell surface. Antibodies that hybridizers or kongugiruut with heterologous polypeptides, also can be used in immunostimulant in vitro and cleanup methods, using methods known in this field. See, for example, WO 93/21232; EP 439095; Naramura et, Immunol. Lett. 39, 1994, cc.91-99; US 5474981; Gillies et, PNAS 89, 1992, cc.1428-1432; and Fell et, J. Immunol. 146, 1991, cc.2446-2452, the essence of which is included in the present invention in the form of links.

Additional hybrid proteins can be obtained by gene shuffling, shuffling motives, shuffling of exons and/or shuffling codons (all of these methods are referred to as "DNA shuffling"). DNA shuffling can be applied to change the action of the antibodies of the present invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation degree). Cm. main publications: US 5605793; 5811238; 5830721; 5834252; 5837458; Patten, etc., Curr. Opinion Biotechnol. 8, 1997, cc.724-733; Harayama, Trends Biotechnol. 16(2), 1998, cc.76-82; Hansson and others, J. Mol. Biol. 287, 1999, cc.265-276; Lorenzo and Blasco, Biotechniques 24(2), 1998, cc.308-313 (the essence of each of these patents and publications are incorporated into this invention by reference). Antibodies or their fragments, or the encoded antibodies or fragments thereof, can be modified the way� random mutagenesis by PCR random errors, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding the antibody or antibody fragment, which specifically bind to IFNAR1, may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Furthermore, antibodies or fragments thereof can gibridizatsiya with marker sequences, such as peptide, to facilitate purification. In other embodiments of the present invention, the marker amino acid sequence is a peptide hexastylis, for example, the label supplied in the vector pQE (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. Description Gentz, etc., Proc. Natl, Acad. Sci. USA 86, 1989, cc.821-824, for instance, hexastylis provides for ease of purification of the hybrid protein. Other peptide tags, applicable for purification include, but not limited to, the label of the hemagglutinin "HA (hemagglutinin), which corresponds to an epitope derived from the hemagglutinin protein of influenza virus (Wilson, etc., Cell 37, 1984, p. 767), and the label "Flag".

In other embodiments of the present invention the antibodies of the present invention, or fragments or derivatives thereof to�nyugyo with diagnostic or detectable agent. Such antibodies can be useful for monitoring or predicting the development or progression of inflammatory disorders as part of a clinical testing method, for example, determining the effectiveness of a specific therapy. Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/Biotin and avidin/Biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorofluorescein, dansyl chloride or phycoerythrine; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and equalin; radioactive materials, such as but their list is not limited to, iodine (131I,125I,123I,121I), carbon (14C), sulfur (35S), riti (3H), indium (115In113In112In111In), technetium (99Tc), thallium (201Ti), gallium (68Ga67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Heh), ft�p ( 18F),153Sm177Lu,159Gd149Pm,140La,175Yb,166Ho90Y47Sc,186Re,188Re,142Pr105Rh,97EN,68Ge57Co,62Zn,85Sr,32P,153Gd169Yb,51Cr54Mn75Se113Sn and117Tin; metals with the positron emission, using various positron-emission tomography, non-radioactive paramagnetic ions of metals and molecules, which are marked with radiometal or anywhereman with certain radioisotopes.

Known methods of conjugation of therapeutic parts of the molecules with antibodies, see, for example, kN.: Arnon et al. "Monoclonal Antibodies and Cancer Therapy, 1985, ed. by Reisfeld, etc., ed. Alan R. Liss, Inc., cc.243-256; kN.: "Controlled Drug Delivery", 1987, ed. by Robinson, etc., 2nd ed., ed. Marcel Dekker, Inc., cc.623-653; kN.: "Monoclonal Antibodies 84: Biological and Clinical Applications", 1985, ed. by Pinchera, etc., cc.475-506; "Monoclonal Antibodies For Cancer Detection and Therapy", 1985, ed. by Baldwin and others, ed. Academic Press, cc.303-316; Thorpe, etc., Immunol. Rev. 62, 1982, cc.119-158.

In another embodiment, the antibody may be anywhereman with a second antibody to form heteroconjugate antibodies as described in the patent US 4676980, the essence of which is included in the present invention by reference. therapeutic part of the molecule or drug anywhereman with the antibody or its fragment that specifically binds to IFNAR1, can be selected for up to�reaches the desired prophylactic or therapeutic effect (effects) in relation to a particular disorder in a subject. A doctor or other medical personnel should consider the following when deciding on what therapeutic part of the molecule or the drug should konjugierte with the antibody or its fragment that specifically bind to IFNAR1: the nature of the disease, the severity of the disease and the condition of the subject.

Methods for obtaining antibodies

Antibodies or fragments thereof can be obtained by any method known in this field, for the synthesis of antibodies, in particular, by chemical synthesis or methods of recombinant expression.

Monoclonal antibodies can be obtained using a wide range of methods known in this field, including the application of a hybrid, recombinant and phage display, or combinations thereof. For example, monoclonal antibodies can be obtained using methods with the use of a hybrid, including known in the field methods and recommendations described, for example, in the book: Harlow et al., "Antibodies: A Laboratory Manual", 1988, publishing Cold Spring Harbor Laboratory Press, 2nd ed.; in the book: Hammerling et al. "Monoclonal Antibodies and T-Cell Hybridomas", 1981, publ Elsevier, new York, cc.563-681 (the essence of these works is included in the present invention in the form of links). The term "monoclonal antibody" as used in the present invention is not limited to the antibodies obtained with the use of a hybrid. The term "monoclonal antibodies�about" refers to the antibody, derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, but not to the method by which it is obtained.

Methods of producing and screening for specific antibodies using a hybrid method, are conventional, well known in this field. Briefly, mice can be immunized IFNAR1, and if an immune response is detected, e.g., antibodies, specific against IFNAR1 detected in serum, get the mouse spleen and isolated from splenocytes. Then splenocytes hybridizer well-known methods, with any appropriate myeloma cells, for example cells from cell line SP20, which can be obtained from the collection of ATCS. The hybridomas are selected and cloned by serial dilutions. Then the hybrid clones are exploring methods known in this field for cells that secrete antibodies capable of binding the polypeptide of the present invention. Ascites fluid, which generally contains high levels of antibodies, can be obtained by immunization of mice positive hybrid clones.

Thus, monoclonal antibodies can be produced by culturing hybrid cells secreting antibody of the present invention, and of hybridomas obtained by fusion of splenocytes isolated from mice immunized with IFNAR1, cell�and myeloma and then screening of hybridomas the resulting hybridization of hybrid clones that secureroot antibody capable of binding IFNAR1.

Fragments of antibodies that recognize specific epitopes IFNAR1, can be obtained by any of the methods known to experts in this field. For example, Fab fragments and F(ab')2the present invention can be obtained by proteolytic cleavage of immunoglobulin molecules, using enzymes, for example papain (Fab fragments) or pepsin (to obtain fragments F(ab')2). Fragments F(ab')2contain the variable region, constant region of light chain and CH1 domain of the heavy chain. In addition, the antibodies of the present invention can also be obtained using various phage display methods known in this field.

In phage display methods, the functional domains of the antibody exhibited on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH domains and VL, amplificateur from cDNA libraries of animals (for example, libraries of lymphoid tissues cDNA of a human or a rodent). DNA encoding the VH domains and VL, recombined together with an scFv linker by PCR and cloned in formigny vector (e.g., pCANTAB 6 or pComb 3 HSS). Vector electroporation in E. coli, and E. coli inficirovannym the phage. Phages used in these methods are typically filamentous phage including fd and M13 and the VH domains and VL is typically recombinante hybridisierung or rahovym gene III, or rahovym genome VIII. Phage expressing antigen-binding domain that binds with the target epitope of IFNAR1, can be selected or identified by the antigen, e.g., using labeled antigen or antigen bound or captured by a solid surface or bead. Examples of phage display methods that can be used to obtain the antibodies of the present invention include the methods described in the work of Brinkman and others, J. Immunol. Methods 182, 1995, SS.41-50; Ames and others, J. Immunol. Methods 184, 1995, SS.177-186; Kettleborough et, Eur. J. Immunol. 24, 1994, SS.952-958; Persic, etc., Gene 187, 1997, pp..9-18; Burton and others, Advances in Immunology 57, 1994, SS.191-280; PCT/GB91/01134; WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and W097/13844; US 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743 and 5969108; the essence of each of these activities is included in the present invention by reference.

According to the above references, after the selection of the phage antibody coding region of the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells insect cells, plant cells, yeast and bacteria, for example, as described below. Methods recombinante received fragments Fab, Fab' and F(ab')2 can also be applied using methods known in this field, for example, described in international publication WO 92/22324; Mullinax et, BioTechniques 12(6), 1992, cc.864-869; Sawai, etc., AJRI 34, 1995, cc.26-34; and Better et, Science 240, 1988, cc.1041-1043 (these works are included in this description by reference in their entity).

To obtain the entire antibody can be applied PCR primers, including the nucleotide sequence of VH or VL, the restriction site, and a flanking sequence to protect the restriction site, for amplification of sequences of the VH or VL in the scFv clones. Using cloning techniques known to experts in this field, the PCR amplified VH domains can be cloned into vectors expressing a constant region VH, for example, the constant region of the gamma 4, and the PCR amplified VL domains can be cloned into vectors expressing a constant region VL, for example, the constant region of the Kappa and lambda man. In some embodiments, the implementation of the present invention, the expression vectors of VH domains or VL comprise the promoter EF-1 alpha secretion signal, a cloning site for the variable domain, constant domain and a selective marker, e.g., neomycin. D�barter VH and VL can also be cloned into the vector, expressing the necessary constant region. The vectors of the conversion of the heavy chain vectors and conversion of light chain then together transferout in cell lines to generate stable or temporary cell lines, which Express the full length antibody, e.g., IgG, using techniques known to experts in this field.

For some applications, including the application in vivo of antibodies to human and methods of detection in vitro, it may be useful to use human antibodies or chimeric antibodies. The antibodies are fully human antibodies, are particularly desirable for therapeutic treatment of humans. Antibodies can be obtained using various methods known in this field, including phage display methods described above using libraries of antibodies derived from sequences of human immunoglobulin. Cm. also US 4444887 and 4716111, international publication WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, each of which is included in the present invention in reference to its essence.

A chimeric antibody is a molecule in which different parts of an antibody derived from different immunoglobulin molecules. Methods of producing chimeric antibodies are known in this field. See, for example, Morrison, Science 229, 1985, p. 1202; Oi et, BioTechniques 4, 1986, p. 214; Gillies et, J. Immunol. Methods 125, 1989, cc.191-202; US 580715, 4816567, 4816397 and 6311415, each of which is included in the present invention in reference to its essence.

Humanized antibody is an antibody or a fragment thereof that is capable of contacting a pre-specific antigen and which includes a frame section having generally the amino acid sequence of a human immunoglobulin and a CDR region amino acid sequence which essentially is a sequence of human immunoglobulin. A humanized antibody comprises substantially all of at least one, and typically two variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or almost all regions correspond to the CDR domains of immunoglobulin, non-human immunoglobulin (i.e., antibody donors), and all or nearly all of the frame sections from a consensus sequence of human immunoglobulin. In some cases, a humanized antibody also comprises at least a portion of constant region of immunoglobulin (Fc), typically of a human immunoglobulin. Typically, the antibody can contain a light chain, and at least the variable domain of the heavy chain. The antibody also may include the CH1, hinge region, CH2, CH3 and CH4 region of the heavy chain. A humanized antibody can be selected from any class of immunoglobulins, including Ig, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. Usually the constant domain is a complement-fixing constant domain, if you want a humanized antibody showed: cytotoxic effect and the class is typically IgG1. If cytotoxic activity is not desirable, the constant domain may be of class IgG2. A humanized antibody may include sequences from more than one class or isotype, and selecting certain constant domains to optimize desired effector functions may be carried out by specialists in this field. Frame portion and the CDR of the humanized antibody does not need to exactly match the original sequence, for example, in CDR consensus donor or frame the site, can be produced by mutations substitutions, insertions or deletions of at least one residue so that the CDR residue or wireframe of the site on this site does not conform to either the consensus or the import antibody. Such mutations, however, may be small. Usually, at least 75% of the humanized antibody residues may correspond to remnants of the original frame region (FR) and CDR sequences, more often 90%, and probably more than 95%. A humanized antibody can be produced using a different method�, known in this area, including, but not limited to, CDR-transplantation (EP 239400, WO 91/09967, US 5225539, 5530101 and 5585089), veneer or update (EP and EP 592106 519596; Padlan, Molecular Immunology 28(4/5), 1991, cc.489-498; Studnicka et, Protein Engineering 7(6), 1994, cc.805-814; Roguska et, PNAS 91, 1994, cc.969-973), chain shuffling (US 5565332) and the methods described, for example, in US 6407213, 5766886, WO 9317105, Tan, etc., J. Immunol. 169, 2002, cc.1119-1125 (2002), Caldas, etc., Protein Eng. 13(5), 2000, cc.353-360, Morea, etc., Methods 20(3), 2000, cc.267-279, Vasa, etc., J. Biol. Chem. 272(16), 1997, cc.10678-10684, Roguska et, Protein Eng. 9(10), 1996, cc.895-904, Couto and others, Cancer Res. 55(23), 1995, cc.5973s-5977s, Couto and others, Cancer Res. 55(8): 1995, cc.1717-1722, Sandhu J. S., Gene 150(2), 1994, cc.409-410, Pedersen, etc., J. Mol. Biol. 235(3), 1994, cc.959-973. Often frame remains in the frame portions may be substituted with the corresponding residue from the CDR donor antibody to modify or improve binding to the antigen. Such substitution in frame areas identified by methods known in this field, e.g., by modeling of the interactions of residues of CDR and framework of the site to identify residues skeleton of the plot, important for antigen binding and sequence comparison to identify unusual balances the frame section according to certain provisions. (See, for example, US 5585089; and Riechmann et, Nature 332, 1988, p. 323, the essence of which is included in the present invention in the form of links).

Polynucleotides that encode an antibody

The present invention also covers the�et polynucleotides which gibridizatsiya in terms of high stiffness, medium or low hardness, for example, as indicated above, with polynucleotide that encode an antibody of the present invention.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides may be determined by any method known in this field. Because the known amino acid sequence antibodies, nucleotide sequences encoding these antibodies can be determined by methods known in this field, i.e., nucleotide codons known to which amino acids they encode, assemble in such a way as to obtain a nucleic acid which encodes the antibody or a fragment thereof of the present invention. Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described Kutmejer, etc., BioTechniques 17, 1994, p. 242), method, which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of these oligonucleotides, and then amplification legirovannykh oligonucleotides by PCR.

In another embodiment, the polynucleotide encoding the antibody may be generated from nucleic acid from a corresponding source. If you clone the contents�schy nucleic acid, which encodes a specific antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from the appropriate source (e.g., a cDNA library of antibodies, or generated from cDNA library, or from nucleic acid, usually poly A+RNA, isolated from, any tissue or cells expressing the antibody, for example, hybrid cells selected for expression of the antibodies of the present invention) by PCR amplification using synthetic primers gibridizatsiya with 3' and 5' ends of the sequence or by cloning using oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may be cloned into replicable cloning vectors using any method known in this field.

If the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be modified by methods known in this field for the manipulation of nucleotide sequences, for example, methods of recombinant DNA, site-directed mutagenesis,PCR, etc. (see, for example, the methods described in the book: Sambrook et Molecular Cloning, A Laboratory Manual", 1990, 2nd ed., publishing Cold Spring Harbor Laboratory, Cold Spring Harbor, new York, and in the book.; "Current Protocols in Molecular Biology", 1998, ed. by Ausubel, etc., the publishing house of John Wiley & Sons, new York, the essence of which is included in the present invention in the form of links) for the development of antibodies with different amino acid sequence, for example, to create amino acid substitutions, deletions and/or insertions.

In one of the embodiments of the present invention, one or more CDR regions embedded in a frame region using conventional methods of DNA recombination. Frame region can be natural or consensual frame sections and, in certain circumstances, skeleton plots (see, for example, the list of frame areas of a person in the work of Chothia et, J. Mol. Biol. 278, 1998, 457-479). Optionally, a polynucleotide obtained by a combination of frame regions and CDRs encodes an antibody that specifically binds to IFNAR1. Optional can be obtained one or more amino acid substitutions in the frame sections, and in some cases amino acid substitution improves the binding of an antibody to its antigen. In addition, such methods can be used to obtain amino acid substitutions or deletions of one or more cysteine residues in variable �area, participating in vnutrisutochnoi disulfide bonds to obtain antibody molecules that have lost one or more vnutrisemejnyh disulfide bonds. Other changes to the polynucleotides of the present invention belong to the scope of the present invention and known to specialists in this field.

In some embodiments of the present invention the antibodies of the present invention encoded by the polynucleotide sequences shown in Fig.1-4. In other embodiments, implementation of the present invention, the polynucleotides of the present invention encode antibodies comprising constant region light chain and heavy chain corresponding to SEQ ID Nos: 41 and 42, respectively. In other embodiments, implementation of the present invention, the polynucleotides of the present invention encode antibodies comprising constant region of the heavy chain corresponding to SEQ ID No:42 with regard to allelic variation, and variation is at least one or more residues selected from the group consisting of provisions, 214, 221, 356 and 358 defined by the numbering system of the EU index.

Recombinant expression of antibodies

Recombinant expression of an antibody of the present invention, derivative, analog or fragment (e.g., a heavy or light chain antibodies by infusion�him to the invention, or part thereof, or single-chain antibodies of the present invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. If you get the polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or a portion (but not necessarily containing the variable domain of the heavy or light chain), according to the present invention, the vector to obtain the antibody molecule can be obtained by recombination of DNA using techniques known in this field. Thus, methods of producing the protein by expression of a polynucleotide containing an antibody encoded by the nucleotide sequence described in the present invention. Methods known to specialists in this field can be used to construct expression vectors containing coding the antibody sequence and the corresponding signals of the control of transcription and translation. These methods include, for example, methods of DNA recombination in vitro, methods of synthesis and genetic recombination in vivo. The present invention, thus, provides replicable vectors comprising a nucleotide sequence encoding the antibody molecule of the present invention, a heavy or light chain antibody variable domain of a heavy or light chain of the antibody or its h�STI, or CDR of the heavy or light chain, functionally associated with the promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., WO 86/05807; WO 89/01036; US 5122464) and the variable domain of the antibody may be cloned into such a vector for expression of a heavy chain, a light chain or a heavy chain and a light chain.

The expression vector is transferred into a host cell by conventional methods, and the transfected cells are then cultured by conventional methods for obtaining antibodies of the present invention. Thus, the present invention includes the host cell containing the polynucleotide encoding the antibody of the present invention or its fragments, or its heavy or light chain, or portion thereof, or single-chain antibody of the present invention, functionally associated with a heterologous promoter. In other embodiments of the present invention for the expression of double-stranded antibodies, vectors encoding both heavy, and light chain, can co-expressed in the cells of the host for expression of the entire immunoglobulin molecule, as detailed below.

Different systems of expression vector can be used for expression of antibody molecules of the present invention (see, e.g., US 5807715). Such with�expression system host represent vectors, which investigated the encoding sequences can be obtained and substantially purified, but also represent cells which may, when transformed or transfection with the respective coding sequences to Express the antibody molecule of the present invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with expression vectors DNA recombinant bacteriophage, plasmid DNA or kosmidou DNA containing the sequence encoding the antibody; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the sequence encoding the antibody; system of insect cells infected with the recombinant expression vectors of the virus (e.g., baculovirus) containing the sequences encoding the antibody; systems of plant cells infected with the recombinant expression vectors of viruses (for example, the cauliflower mosaic virus (cauliflower mosaic virus - CaMV), tobacco mosaic virus (tobacco mosaic virus - TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing sequences encoding the antibody; or system of mammalian cells (e.g., COS cells, Cho, BHK, 293,NS0, and 3T3), supporting structures of recombinant expression containing promoters derived from the genome of mammalian cells (for example, the promoter of metallothionein) or from mammalian viruses (e.g., adenovirus late promoter; the promoter of vaccinia virus 7.5 K). In some embodiments of the present invention, a bacterial cell such as Escherichia coli, and in other embodiments of the present invention, eukaryotic cells, especially for the expression of whole recombinant molecules antibodies used for expression of recombinant molecules of the antibody. For example, mammalian cells, e.g., cells of the Chinese hamster ovary (Chinese hamster ovary cells - SSS), in the clutch with the vector, for example, the main element of the intermediate early gene promoter of human cytomegalovirus are an effective expression system antibodies (Foecking et, Gene 45, 1986, p. 101; and Cockett et, Bio/Technology 8, 1990, p. 2). In some embodiments of the present invention, the expression of nucleotide sequences encoding antibodies or fragments thereof that specifically bind to IFNAR1, is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems mainly can be selected a number of expression vectors, depending on the application �distributions of antibody molecules, intended for expression. For example, when you receive a large amount of protein for the generation of pharmaceutical compositions of an antibody molecule can be desirable vectors directing the expression of high levels of products of hybrid proteins that are easy to clean. Such vectors include, but is not limited to, the expression vector E. coli pUR278 (Ruther et, EMBO 12, 1983, p. 1791), which encodes the antibody sequence can be Legerova into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye &Inouye, Nucleic Acids Res. 13? 1985, cc.3101-3109; Van Heeke &Schuster, J. Biol. Chem. 24, 1989, cc.5503-5509); and the like. pGEX Vectors may also be used for expression of foreign polypeptides as hybrid proteins with glutathione-5-transferase (glutathione 5-transferase - GST). In General, such hybrid proteins are soluble and can easily be purified from lidirovavshy cells by adsorption and binding to matrix glutathione agarose pellets, followed by elution in the presence of free glutathione. The pGEX vectors are designed to include cleavage sites of thrombin or protease factor XA in such a way that the product can be released from the GST molecules.

In the cells of the host mammal can be used a number of expression systems based on viruses. In cases where adenov�Rus is used as an expression vector, investigated encoding the antibody sequence can be ligitamate with a control complex transcription/translation of adenovirus, e.g., the late promoter and tripartite leader sequence. This chimeric gene could then be insertion in the genome by recombination in vitro or in vivo. Insertion in a small region of the viral genome (e.g., region E1 or E3) may result in a recombinant virus that is viable and can Express the antibody molecule in infected hosts (e.g., see Logan and Shenk, Proc. Natl. Acad. Sci. USA 81, 1984, cc.355-359). Specific initiation signals may also be required for efficient translation of coding sequences insertional antibodies. Such signals include the initiation codon ATG and related sequences. In addition, the initiating codon must be in phase with the reading frame of the desired coded sequence to ensure translation of the entire insert. Such exogenous signals control the broadcast and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of relevant elements enhance transcription, transcription terminators, etc. (see, e.g., Bittner et, Methods in Enzymol. 153, 1987, cc.516-544).

Chrome�, can be a selected strain of host cells, which modulates the expression of insertionindex sequences, or modifies and processorwith specific gene product in the required manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different cells-the owners have properties and characteristic mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host system can be selected to ensure appropriate modification and processing of alien expressed protein. This can be done in eukaryotic cells-the owners of which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such cells-the owners are, but not limited to, cells of Cho, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, VT, Hs578T, HTB2, BT2O and T47D, NS0 (line myeloma cells of rodent, in which endogenous does not produce any immunoglobulin chains), CRL7O3O and HsS78Bst.

For a long period at a high level production of recombinant proteins preferred stable expression. For example, can be engineered cell line, which stably Express the antibody molecule. Prima�structure of expression vectors, which contain a viral origin of replication is preferred if the host cell can be transformed with DNA controlled by appropriate control elements of expression (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selective marker. After the introduction of alien DNA, engineered cells can grow for 1-2 days in an enriched media, and then they are transferred to a selective medium. The selective marker in the recombinant plasmid causes resistance to the selection and allows cells stable integration of the plasmid into the chromosome and growth for the formation of the center, which in turn can be cloned and propagated in cell lines. This method may advantageously be used to construct cell lines expressing the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that directly or indirectly interact with a molecule of the antibody.

Can be used a number of systems selection, including, but not limited to, genes timedancing virus herpes simplex (Wigler et, Cell 11, 1977, p. 223), (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48, 1992, p. 202) and adrinfo.standortstr (Lowy et, Cell 2, 1980, cc.8-17), in tk-, hgprt - or aprt - cells, respectively. Can also be used resistance to antimetabolites as the basis for the selection of the following genes: dhfr gene, which conditions resistance to methotrexate (Wigler, etc., Natl. Acad. Sci. USA 77, 1980, p. 357; O'hare, etc., Proc. Natl. Acad. Sci. USA 78, 1981, p. 1527); gpt gene, which conditions resistance to mycophenolic acid (Mulligan &Berg, Proc. Natl. Acad. Sci. USA 78, 1981, p. 2072); neo gene, which conditions resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3, 1991, cc.87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32, 1993, cc.573-596; Mulligan, Science 260, 1993, cc.926-932; Morgan and Anderson, Ann. Rev. Biochem. 62, 1993, cc.191-217; May, TIB TECH 11(5), 1993, cc.155-215); and hygro gene, which conditions resistance to hygromycin (Santerre, etc., Gene 30, 1984, p. 147). Methods well known in the field of recombinant DNA, can be properly applied for the selection of the desired recombinant clone; they are described, for example, in the book: "Current Protocols in Molecular Biology", 1993, ed. by Ausubel and others, ed. John Wiley & Sons, new York; in the book: Kriegler "Gene Transfer and Expression, A Laboratory Manual", 1990, ed. Stockton Press, NY; in proc.: "Current Protocols in Human Genetics", 1994, edited by Dracopoli, etc., ed. John Wiley & Sons, new York, chapters 12 and 13; Colberre-Garapin, etc., J. Mol. Biol. 150, 1981, p. 1; these works included in the present invention in the form of links to their essence.

The expression levels of an antibody molecule can be increased by amplification of the vector (see the review in the book: Bebbington and Hentschel, "The use of vectors based on gene amplificaion for the expression of cloned genes in mammalian cells in DNA cloning", 1987, ed. Academic Press, new York, vol. 3). If the marker in the vector system expressing antibody is amenable to amplification, increasing the level of inhibitor contained in the culture of host cells may increase the number of copies of a gene marker. Because amplificatory region associated with the antibody gene, production of the antibody may also increase (Crouse et, Mol. Cell. Biol. 3, 1983, p. 257).

A host can be co-transfected with two expression vectors of the present invention: the first vector encodes a polypeptide that is derived from the heavy chain and the second vector encodes a polypeptide that is derived from the light chain. Two vectors can contain identical selective markers that show equal expression of the polypeptides of the heavy and light chains. In another embodiment, may also be one vector which encodes and is capable of expressing polypeptides and heavy and light chains. In such cases, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322, 1986, p. 52; Kohler, Proc. Natl. Acad. Sci. USA 77, 1980, 2, p. 197). Coding sequences of the heavy and light chains may include cDNA or genomic DNA.

If the antibody molecule of the present invention is obtained by recombinant expression, they can clear any method known in this field for �cleaning of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly affine in relation to the specific antigen after protein A, and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique intended for protein purification. In addition, the antibodies of the present invention or their fragments can be hybridisierung with heterologous polypeptide sequences described in the present invention, or other known in this area, which can facilitate cleaning.

Scaling generate antibodies

Large quantities of antibodies of the present invention can be obtained by using a scalable process (referred to in the present invention the scalable process of the present invention"), In some embodiments of the present invention antibodies can be obtained by a scalable process of the present invention in the research laboratory that may be scaled to obtain the antibodies of the present invention in analytical scale bioreactors (for example, but not limited to, in bioreactors with a volume of 5 l, 10 l, 15 l, 30 l or 50 l). In other embodiments of the present invention antibodies can be obtained using masstube�creating process according to the present invention in the research laboratory, in the course of which can be enhanced to develop to the level of production of the antibody of the present invention in industrial scale bioreactors (for example, but not limited to, 75 l, 100 l, 150 l, 300 l or 500 l). In some embodiments of the present invention, the scalable process of the present invention causes little or unmodified production efficiency compared drafting process carried out in a research lab. In other embodiments of the present invention, a scalable method of the present invention allows to obtain antibodies with generation efficiency at the level of about 10 mg/l, about 20 mg/l, about 30 mg/l, about 50 mg/l, about 75 mg/l, about 100 mg/l, about 125 mg/l, about 150 mg/l, about 175 mg/l, about 200 mg/l, about 250mg/l, about 300 mg/l or higher. In other embodiments of the present invention, the hybrid protein can be obtained by scaling the methods of the present invention.

In other embodiments of the present invention, a scalable method of the present invention allows to obtain antibodies with generation efficiency at the level of at least about 10 mg/l, at least about 20 mg/l, at least about 30 mg/l, at least about 50 mg/l, at moreprepare 75 mg/l, at least about 100 mg/l, at least about 125 mg/l, at least about 150 mg/l, at least about 175 mg/l, at least about 200 mg/l, at least about 250 mg/l, at least about 300 mg/l or more.

In other embodiments of the present invention, a scalable method of the present invention allows to obtain antibodies with generation efficiency at the level of at least about 10 mg/l to about 300 mg/l, from about 10 mg/l to about 250 mg/l, from about 10 mg/l to about 200 mg/l, from about 10 mg/l to about 175 mg/l, from about 10 mg/l to about 150 mg/l, from about 10 mg/l to about 100 mg/l, from about 20 mg/l to about 300 mg/l, from about 20 mg/l to about 250 mg/l, from about 20 mg/l to about 200 mg/l 20 mg/l to about 175 mg/l, from about 20 mg/l to about 150 mg/l, from about 20 mg/l to about 125 mg/l, from about 20 mg/l to about 100 mg/l, from about 30 mg/l to about 300 mg/l, from about 30 mg/l to about 250 mg/l, from about 30 mg/l to about 200 mg/l, from about 30 mg/l to about 175 mg/l, from about 30 mg/l to about 150 mg/l, from about 30 mg/l to about 125 mg/l, from about 30 mg/l to about 100 mg/l, from about 0 mg/l to about 300 mg/l, from about 50 mg/l to about 250 mg/l, from about 50 mg/l to about 200 mg/l, 50 mg/l to about 175 mg/l, primerno 50 mg/l to about 150 mg/l, from about 50 mg/l to about 125 mg/l, or from about 50 mg/l to about 100 mg/L.

Other ways of constructing antibodies

In another embodiment of the present invention, the hinge region Fc of the antibody of the present invention are mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations introduced in the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has reduced binding of staphylococcal protein A (Staphylococcyl protein A - SpA) compared to the binding of native Fc-hinge domain SpA. This approach is described in more detail in US 6165745.

In another embodiment of the present invention, the antibody is modified to increase the biological half-period of existence. There are various methods. For example, can be implemented by one or more of the following mutations: T252L, T254S, T256F as described in US 6277375. In another embodiment of the present invention may be implemented by one or more of the following mutations: M252Y, S254T, THE described in US 7083784. In another embodiment, to increase biological half-period of existence, the antibody may be modified in the field CH1 or CL for content reutilizing receptor-binding epitope taken from two loops of a CH2 domain about�Asti in the IgG Fc as described in US 5869046 and 6121022.

In other embodiments, implementation of the present invention, the Fc region is modified by substituting at least one amino acid residue for another amino acid residue to reduce the effector function (s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced by other amino acid residues such that the antibody becomes low affinity to the effector ligand but retains the antigen-binding ability of the original antibody. The effector ligand to which affinity is lowered, may be, for example, Fc receptor or the C1 component of complement. This approach is further described in detail in US 5624821 and 5648260.

In another example, one or more amino acid residues selected from amino acid residues 329, 331 and 322 can be replaced by other amino acid residues such that the antibody becomes reduced C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in more detail in US 6194551.

In another example, one or more amino acid residues at the positions of amino acids 231 and 239 modified, so as to reduce the ability of the antibody to fix complement. This approach is further described � PCT publication WO 94/29351.

In still another embodiment of the present invention, the Fc region of the antibody of the present invention is additionally modified to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of an antibody to an Fey receptor by modifying one or more amino acids in the following provisions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is further described in PCT publication WO 00/42072.

Another modification of the antibodies in the context of the present invention is achieved by paglinawan. The antibody may be paglinawan, for example, to increase the biological half-period of the existence of antibodies (e.g., in serum). Paglinawan antibody or its fragment is usually achieved by reaction with polyethylene glycol (PEG), for example, with a reactive ester or aldehyde derivative of PEG, under conditions in which one or more groups of the PEG become attached to the antibody or antibody fragment. In some cases, paglinawan is carried out via reaction of atsilirovaniya or an alkylation reaction with a reactive molecule of PEG (or similar water-soluble floor�measure). In the context of the present invention, the term "polyethylene glycol" encompasses any of the forms of PEG that is used to produce derivatives of other proteins, for example, mono(C1-C10)alkoxy - or aryloxypropanolamine or polietilenglikolsuktsinata. In some embodiments of the present invention the antibody is designed to paglinawan is glycosylamines antibody. Methods paglinawan proteins known in this field and can be applied to the antibodies of the present invention. See, for example, EP 0154316 and EP 0401384.

Thus, in another object of the present invention structural properties of anti-IFNAR1 antibodies, for example, 3F11, 4G5, E and 9D4, but their list is not limited to, are used to create structurally related anti-IFNAR1 antibody that retains at least one functional property of the antibodies of the present invention, such as binding to IFNAR1. For example, one or more CDR regions of antibodies 3F11, 4G5, E or 9D4, or their mutations can be recombinante combined with known frame areas and/or other areas of CDRs to create additional recombinante designed anti-IFNAR1 antibodies of the present invention described above. Other types of modifications include modifications described in the previous section. The original Mat�the chart for a method of engineering one or more sequences of V Hand/or VLdescribed in the present invention, or one or more of their CDR regions. To create the engineered antibody is not necessary really to receive (i.e. to Express in the form of a protein) an antibody having one or more sequences of VHand/or VLcontemplated in the present invention, or one or more of their CDR regions. Preferably the information contained in a sequence (sequence), are used as source material for creating sequences (sequences) of the "second generation", derived from the original sequence (of sequences), and then the sequence (s) "second generation" prepare and Express as a protein.

Composition

In another embodiment of the present invention provide for compositions containing one monoclonal antibody or a combination of monoclonal antibodies or hybrid proteins comprising the Fc region, according to the description of the present invention, recycled together with the carrier. Such compositions can include one antibody or a combination (e.g., two or more different) antibodies, hybrid proteins, immunoconjugate or bespecifically molecules of the present invention. In some embodiments, at�present invention such compositions are physiologically tolerated and therefore applicable for administration to a subject (also referred to as "pharmaceutical compositions of the present invention"). For example, pharmaceutical compositions of the present invention may include a combination of antibodies (or immunoconjugates, or bespecifically antibodies) that bind to different epitopes on the antigen target, or which have a complementary effect.

In another embodiment of the present invention the compositions of the present invention can include one or more pharmaceutically acceptable salts. Examples of such salts include acid additive salts and basic additive salt. To the acid additive salts include non-toxic salts of inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, methyl-hydrogen, iodine and hydrogen, phosphorous, etc., salts of non-toxic organic acids such as aliphatic mono - and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoate acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. To the basic additive salts include salts of alkali and alkaline earth metals, e.g. sodium, potassium, magnesium, calcium, etc., as well as nontoxic organic amines, e.g. N,N'-dibenziletilendiaminom, N-methylglucamine, chloroprocaine, choline, diethanolamine, Ethylenediamine, procaine and the like.

In another embodiment of the present invention the compositions according to the infusion�him to the invention can also include pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (butylated hydroxyanisole - BHA), butylated hydroxytoluene (butylated hydroxytoluene - BHT), lecithin, propylgallate, alpha-tocopherol, etc.; and (3) the agents chelate metals, for example, citric acid, ethylenediaminetetraacetic acid (ethylenediamine tetraacetic acid - EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

Examples of appropriate aqueous and non-aqueous media, which can be used in the described compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and appropriate mixtures thereof, vegetable oils, e.g., olive oil, and injectable organic esters, for example, ethyloleate. The desired fluidity can be maintained, for example, the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surface active agents.

In another embodiment of the present invention the compositions of the present invention may also include� adjuvants, for example, preservatives, wetting agents, emulsifying agents and dispersing agents. To prevent the ingress of microorganisms can or sterilization, or the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, menolascino acid, etc. can Also be it is desirable to include isotonic agents, for example Sugars, sodium chloride, etc., in the composition. In addition, prolonged absorption of the injectable pharmaceutical form may be effected, for example, by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

To pharmaceutically acceptable agents include sterile aqueous solutions or dispersions and sterile powders for fast preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in this field. The exception is any conventional media or agent, associated with the current connection, the application of which is considered in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be included in the composition. Therapeutic compositions typically must be sterile and stable under the conditions of preparation and storage. Composition �can be recycled in the form of a solution, microemulsions, liposomes or other structure, is applicable for high concentration of the drug. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol, etc.) and their respective mixtures. The necessary fluidity can be maintained, for example, by applying a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surface active agents. In many cases it may be useful for inclusion isotonic agents, for example, Sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride in the composition. In addition, prolonged absorption of the injectable compositions can be carried out, for example, the inclusion in the composition of an agent that delays absorption, for example, monostearate and gelatin.

Sterile injectable solutions can be prepared by inclusion of active compounds in the required amount in an appropriate solvent, alone or in combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Typically, dispersions are prepared by the incorporation of active compounds in sterile solvent, which contains primary�Yu dispersion medium and the required other ingredients listed above. In the case of sterile powders for obtaining sterile injectable solutions, the preferred methods of obtaining are vacuum drying and lyophilization, which produce a powder of an active ingredient plus any additional desired ingredient from a previously sterilized by filtration of the solution.

In one embodiment of the present invention, compositions (e.g., liquid formulations) of the present invention are compositions that do not contain pyrogens, which are substantially free of endotoxins and/or loved ones of pyrogenic substances. The endotoxins are toxins that are present inside the microorganisms and is released with the destruction or death of microorganisms. Pyrogenic substances also include inducing fever thermostable substances (glycoproteins) from the outer membrane of the bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock when administered to humans. Because of the potentially harmful effects it is useful to remove even small amounts of endotoxins of injectable pharmaceutical solutions. Management on control over foodstuff and medicines of the USA (The Food & Drug Administration - FDA) has set an upper limit of 5 units of endotoxin (endotoxin units, EU) per dose per kg of body weight p�ateenyi one hour of intravenous administration of the drug (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1), 2000, p. 223). With the introduction of therapeutic proteins in quantities of several hundred or thousand mg, for example, in the case of monoclonal antibodies, it is useful to remove even traces of endotoxin. In one embodiment of the present invention, the levels of endotoxin and pyrogene in the composition is less than 10 EU/mg, or less then 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less then 0.01 EU/mg, or less than 0.001 EU/mg. In another embodiment of the present invention, the levels of endotoxin and pyrogene in the composition is less than about 10 EU/mg, or less than about 5 EU/mg, or less than about 1 EU/mg, or less than about 0.1 EU/mg, or less than about 0.01 EU/mg, or less than about of 0.001 EU/mg.

The amount of active ingredient that may be combined with a carrier material to obtain a single metered-dose forms may vary depending on the subject being treated and the mode of introduction. The amount of active ingredient that may be combined with a carrier material to obtain a single metered-dose form, usually can be the amount of the composition that causes therapeutic effect. Usually 100% of this amount can vary from about 0.01% to about 99% active ingredient, also from about 0.1% to about 70%, and from about 1% to about 30% active �of ingredient in combination with a pharmaceutically acceptable carrier.

Dosing regimens set to provide the optimum desired response (e.g., therapeutic response). For example, there may be a single bolus, several individual doses over time or the dose may be proportionally reduced or increased depending on the requirements in relation to the specific therapeutic situation. Especially it is preferable to process a parenteral composition in dosage form for ease of administration and uniformity of dosage. A dosage form in the context of the present invention refers to physically discrete units suitable as single doses for the treatment of subjects; each dosage form contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. Technical requirements for metered-dose dosage forms of the present invention are dictated by and directly dependent on (a) certain properties of the current connection and a specific therapeutic effect, which strive to achieve, and (b) mandatory restrictions in this area the preparation of active compounds for the treatment of sensitivity in individuals.

For the introduction of antibodies dose varied in the range of approximately of 0.0001-100 m�/kg, more usually 0.01 to 5 mg/kg of body weight of the host. For example, dosages can be 0.3 mg/kg of body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg of body weight, or 1-10 mg/kg. a Typical treatment regime enshrines the introduction of once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every 3-6 months. Dosing regimens of the anti-IFNAR1 antibodies of the present invention include 1 mg/kg body weight or 3 mg/kg of body weight, injected intravenously with the antibody used, using one of the following dosing schemes: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) 3 mg/kg of body weight once, and then 1 mg/kg of body weight every three weeks.

In another embodiment, the antibody is a hybrid protein can be introduced as a composition sustained release, and in this case requires less frequent administration. The dosage and frequency of administration depend strongly on the half-life of the antibody in the patient's body. Typically human antibodies show the greatest half-life, followed by humanized antibodies, chimeric antibodies and antibody, non-human antibodies. Dose and frequency of administration can vary depending on whether treatment is prophylactic �whether therapeutic. Prophylactic use of a relatively low dosage is administered at relatively rare intervals over a long period. Some patients receive treatment for the rest of life. In therapeutic applications sometimes require a relatively high dosage at relatively short intervals until such time as the patient may be partial or complete improvement of symptoms. Subsequently, the patient is transferred to a preventive mode.

The actual levels of active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration without the toxicity to the patient. The selected dose level may depend upon a variety of pharmacokinetic factors including the activity of a particular composition used according to the present invention, or the ester, salt or amide, the method of administration, time of administration, the degree of excretion of the specific compounds used, the duration of the treatment, other drugs, compounds and/or materials used in combination with defined compositions used, age, gender, body mass index, comp�States, General health and prior medical history of the patient being treated, and other factors in medicine.

A therapeutically effective dose of anti-IFNAR1 antibodies of the present invention reduces the severity of symptoms, increased frequency and duration of periods of the disease without displaying symptoms or prevention of impairment or disability due to the severity of the symptoms of the disease. In the case of, for example, systemic lupus erythematosus (SLE) a therapeutically effective dose can prevent further deterioration of physical symptoms associated with SLE, for example, pain, fatigue or weakness. A therapeutically effective dose can also prevent or delay SLE that may be desirable in the presence of early or advanced symptoms of the disease. Also, a therapeutically effective dose delays the progression of chronic associated with SLE. Laboratory tests used in the diagnosis of SLE include chemical, hematological, serological and radiological tests. Thus, can be applied to any clinical or biochemical research that allows you to monitor any of the above indicators to determine whether a certain treatment a therapeutically effective dose for the treatment of�I SLE. The person skilled in the art can determine such amounts based on certain factors, such as the size of the subject, the severity of symptoms in a subject, a particular track or the selected method of administration.

The composition of the present invention may be introduced by one or more routes of administration using one or more of various methods known in this field. The experts know that the path and/or the method of administration can vary depending on the desired results. Selected methods of administration of antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, along with other parenteral routes of administration or other parenteral routes of administration, e.g., injection or mixed methods. Parenteral administration may represent a route of administration different from enteral or local administration, usually by injection, and includes, but is not limited to, methods of administration: intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intra-ocular, intracardiac, intradermal, intraperitoneal, nutritherapy, subcutaneous, under the cuticle, intra-articular, podkapsul'nye, subarachnoid, intraspinal, epidural and intramammary and infusion.

In another �the Ariant implementation of the present invention, the antibody of the present invention can be introduced to others, than parenteral, by, for example, topical, epidermal or through a mucous membrane, e.g. intranasally, orally, vaginally, rectally, subligual or topically.

The active compounds can be processed with carriers that will protect the compound against rapid release, such as controlled release, including implants, percutaneous patches, and microencapsulated system of delivery. Can be used biodegradable biocompatible polymers, e.g., vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyarteritis and primelena acid. Many methods of producing such formulations are patented or generally known to specialists in this field. See, for example, kN.: "Sustained and Controlled Release Drug Delivery Systems', 1978, edited by J. R. Robinson, ed. Marcel Dekker, Inc., New York.

Therapeutic compositions can be administered with medical devices known in this field. For example, in another embodiment of the present invention, a therapeutic composition of the present invention may be administered using the device for subcutaneous injection without a needle, for example, using the devices described in US 5399163,5383851, 5312335, 5064413, 4941880, 4790824 or 4596556. Examples of well-known implants and modules applicable in the present invention are described in the patents: US 4487603 that describes them�entirely microinfusion pumps for distribution of the drug at a controlled level; US 4486194, which describes a therapeutic device for administering drugs through the skin; US 4447233, which describes a pump for infusion drugs for delivering medicines with a specific infusion rate; US 4447224, which describes an implantable device for infusion with variable feed rate for continuous injection of the drug; US 4439196, which describes the osmotic drug delivery system with multi-chamber compartments; and US 4475196, which describes the osmotic drug delivery system. These patents included in the present invention in the form of links. Many other such implants, delivery systems, and modules are known to specialists in this field.

In some embodiments of the present invention the antibodies of the present invention can be processed to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) includes many highly hydrophilic compounds. To ensure that therapeutic compounds of the present invention cross the BBB (if necessary), they should be recycled, for example, in liposomes. About methods of obtaining liposomes, see, e.g., US 4522811, 5374548 and 5399331. Liposomes can include one or several parts of molecules that selection� transferred into certain cells or organs thus enhancing targeted drug delivery (see, e.g., V. V. Ranade J. Clin. Pharmacol. 29, 1989, p. 685). Examples of target parts of the molecules are folate or Biotin (see, e.g., US 5416016); mannoside (Umezawa, etc., Biochem. Biophys. Res. Commun. 153, 1988, p. 1038); antibodies (P. O. Bloeman, etc. FEBS Lett. 357, 1995, p. 140; M. Owais, etc. Antimicrob. Agents Chemother. 39, 1995, p. 180); surface-active receptor protein A (Briscoe et Am. J. Physiol. 1233, 1995, p. 134; p120 (Schreier et J. Biol. Chem. 269, 1994, p 9090); see also K. Keinanen; M. L. Laukkanen FEBS Lett. 346, 1994, p. 123; J. J. Killion; I. J. Fidler Immunomethods 4, 1994, p. 273.

The use in diagnostics

In other embodiments of the present invention the antibodies of the present invention are useful as diagnostic and therapeutic agents in vitro and in vivo. For example, these molecules can be introduced into cells in culture, e.g. in vitro or ex vivo, or in the body of a subject, e.g., in vivo, for the treatment, prevention or diagnosis of various diseases.

In one of the embodiments of the present invention the antibodies of the present invention can be applied for the detection of IFNAR1 levels or levels of cells that Express IFNAR1. This can be achieved, for example, by contact of the sample (e.g., sample in vitro) and a control sample with an anti-IFNAR1 antibody under conditions that allow for formation of a complex between the antibody and IFNAR1. Any complexes formed between an�iteam and IFNAR1, detected and compared in the sample and in the control. For example, standard detection methods known in this field, for example, ELISA and liquid cytometry, can be carried out using the compositions of the present invention.

Thus, one object of the present invention also provides methods for detecting the presence of IFNAR1 (e.g., antigen human IFNAR1) in a sample, or measuring the amount of IFNAR1, including contacting the sample and a control sample with the antibody of the present invention or the antigen-binding portions that specifically bind to IFNAR1 under conditions that allow formation of a complex between the antibody or part of it and IFNAR1. Then detect the formation of the complex, wherein a difference in complex formation between the test sample and the control sample is indicative of the presence in the sample IFNAR1.

Therapeutic use

IFNAR1 is part of the cellular receptor for type I interferons, and it is known that type I interferons are immunoregulatory cytokines involved in the differentiation of T cells, production of antibodies and in action or survival of T-cell memory. In addition, increased expression of type I interferons described in many autoimmune diseases, HIV infection, transplant rejection� and disease graft-versus-host (TNX). Thus, the anti-IFNAR1 antibodies of the present invention or fragments thereof, that inhibit the functional activity of type I interferons, can be used in various clinical indications, including aberrant or unwanted action of type I interferons. The present invention encompasses methods of preventing, treating, maintaining, facilitating or suppression mediated by interferon type I diseases or disorders, which are provided for the introduction of antibodies or their antigen-binding portions of the present invention.

Specific examples of autoimmune conditions in which the antibodies of the present invention can be applied include, but is not limited to, the following States of: systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's disease, ulcerative colitis and celiac disease), multiple sclerosis (PC), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis. In addition, the compositions of the antibodies of the present invention can be used for inhibiting or preventing transplant rejection or for the treatment of disease graft-versus-host (TNX) or for the treatment of HIV/AIDS.

High IFNα levels detected in the serum of patients with systemic CRA�Noah erythematosus (SLE) (see, for example, Kim et al. Clin. Exp. Immunol. 70, 1987, cc.562-569). In addition, it is shown that the introduction of IFNα, for example in the treatment of cancer or viral infections, induces SLE (Garcia-Porrua et Clin. Exp. Rheumatol. 16, 1998, cc.107-108). Thus, in another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention can be used for the treatment of SLE by introducing antibodies to a subject in need of such treatment.

Other methods of treatment of SLE are described in patent applications US 60/907767, 60/966174 and PCT/US2007/02494, the essence of each of which are included in the present invention by reference.

IFNα is also involved in the pathology of diabetes of the first type. For example, the presence of immunoreactive IFNα in the beta cells of the pancreas in patients of type I (Foulis et Lancet 2, 1987, cc.1423-1427). It was also shown that prolonged use of IFNα in antiviral therapy induces type I diabetes (Waguri et Diabetes Res. Clin. Pract. 23, 1994, cc.33-36). Thus, in another embodiment of the present invention, the anti-IFNAR1 antibodies or fragments thereof of the present invention can be used for the treatment of type I diabetes by introducing antibodies to a subject in need of such treatment. The antibody can be used alone or in combination with other antidiabetic agents, for example, with insulin.

It is established that antibodies to IFNAR1 effective in animal models with in�politeley bowel disease (see patent application US 60/465155). So anti-IFNAR1 antibodies or fragments thereof of the present invention can be used in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease by introducing antibodies to a subject in need of such treatment.

The IFNα treatment was also investigated for the induction of autoimmune thyroiditis (Monzani et Clin. Exp. Med. 3, 2004, cc.199-210; Prummel and Laurberg Thyroid 13, 2003, cc.547-551). Thus, in another embodiment of the present invention, the anti-IFNAR1 antibodies of the present invention can be used in the treatment of autoimmune thyroid disease, including autoimmune primary hypothyroidism, graves ' disease, Hashimoto's thyroiditis and destructive thyroidism with hipotiroidismo the introduction of the antibodies of the present invention to a subject in need of such treatment. The antibodies of the present invention can be used alone or in combination with other agents or treatments, with anti-thyroid drugs, radioactive iodine and intermediate thyroidectomies.

High levels of IFNα are also observed in the bloodstream of patients with HIV infection, and its presence is a predictor of progression to AIDS (DeStefano et J. Infec. Disease 146, 1982, p. 451; Vadhan-Raj et Cancer Res. 46, 1986, p. 417). Thus, in another embodiment of the infusion�his invention of the anti-IFNAR1 antibodies of the present invention can be used to treat HIV infection or AIDS by administering antibodies of the present invention to a subject, in need of this treatment. In another embodiment of the present invention the antibodies of the present invention may be applied separately or in combination with other anti-HIV agents, for example, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, protease inhibitors and hybrid inhibitors.

It is established that antibodies to IFNAR1 effective in suppressing allograft rejection and prolonging allograft survival (see, e.g., Tovey, etc. J. Leukoc. Biol. 59, 1996, cc.512-517; Benizri et J. Interferon Cytokine Res. 18, 1998, cc.273-284). Thus, the anti-IFNAR1 antibodies of the present invention can also be applied by a recipient of a transplant for inhibiting allograft rejection and/or prolonging survival of an allograft. The present invention provides a method of inhibiting transplant rejection by introducing anti-IFNAR1 antibodies of the present invention to a transplant recipient in need of such treatment. Examples of tissue grafts that can be treated are, but not limited to, liver, lungs, kidneys, heart, small intestine and islet cells of the pancreas, and treatment of the disease graft-versus-host (TNX). The antibodies of the present invention can be applied DEP�flax or in combination with other agents for suppressing transplant rejection, for example, immunosuppression agents (e.g., cyclosporine, azathioprine, methylprednisolone, prednisolone, prednisone, mycophenolate of mofetil, sirolimus, rapamycin, tacrolimus), anti-infective agents (e.g., acyclovir, clotrimazole, ganciclovir, nystatin, trimethoprimsulfamethoxazole), diuretics (e.g., bumetanide, furosemide, metolazone) and medicines to treat ulcers (e.g. cimetidine, famotidine, lansoprazola, omeprazole, ranitidine and sukralfat).

In some embodiments, the present invention provides methods for the introduction and implementation of the compositions and antibodies of the present invention for the treatment and prevention of a wide range of inflammatory conditions, including chronic and acute conditions, such as, but not limited to, appendicitis, peptic ulcers, gastric ulcers and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottis, alagasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, Allergy, anaphylactic shock, immune complex disease, ischemia of organs, reperfusion injury, necrosis, hay fever, sepsis, septicemia, endotoksicski shock, cachexia, hyperpyrexia�, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, pulmonary ultramicroscopic silicic volcanic konis, alveolitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, herpes infection, HIV infection, infection of hepatitis b virus infection hepatitis C virus infection, disseminated bacteremia, Dengue fever, candidiasis, malaria, filariasis, AMIBIOS, alveococcosis, burns, dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals, vasculitis, angitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic attack, Alzheimer's disease, glutencontaining celiac disease, congestive heart failure, restenosis, chronic obstructive pulmonary disease (chronic obstructive pulmonary disease - COPD), syndrome of respiratory disorders in adults, meningitis, encephalitis, multiple sclerosis, ischemic stroke, cerebral embolism, Guillain-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgia, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, asthenic bulbar paralysis, T.�eodit, systemic lupus erythematosus syndrome Goodpasture, 's Behcet's syndrome, allograft rejection, disease graft-versus-host, type I diabetes, ankylosing spondylitis, Berger disease, Reiter's syndrome and Hodgkin's disease.

In another embodiment of the present invention, methods of administration and compositions of the antibodies of the present invention may be useful for the prevention, treatment, relief of symptoms associated with conditions or diseases, including: graves ' disease, Hashimoto's thyroiditis, Crohn's disease, psoriasis, psoriatic arthritis, sympathetic ophthalmia, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome, sjögren syndrome, scleroderma, Addison's disease, polyendocrine syndrome of deficiency, Guillain-Barre syndrome, immune thrombotic purple, pernicious anemia, myasthenia gravis, primary biliary cirrhosis, mixed connective tissue disease, vitiligo, autoimmune uveitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, celiac disease, dermatitis herpetiformis, autoimmune hepatitis, pemphigus, pemphigus vulgaris, pemphigus foliaceous, bullous pemphigoid, autoimmune myocarditis, autoimmune vasculitis, circular alopecia, autoimmu�tion atherosclerosis, Of Behcet's disease, autoimmune myelopathy, autoimmune hemophilia, autoimmune interstitial cystitis, autoimmune diabetes insipidus, autoimmune endometriosis, recurrent polyhedric, ankylosing spondylitis, autoimmune urticaria, dermatomyositis, syndrome of Miller Fisher, Berger disease, goodpasture's syndrome and herpes pregnant women.

In another embodiment of the present invention, methods of administration and compositions of the antibodies of the present invention may be useful for the prevention, treatment, relief of symptoms associated with Sjogren's syndrome. Sjogren's syndrome is an autoimmune disorder in which immune cells overwhelm and destroy the endocrine glands that secrete lacrimal fluid and saliva. The syndrome is named after Swedish ophthalmologist Henrik Sjogren (1899-1986), first described. Sjogren's syndrome are also associated with rheumatic disorders such as rheumatoid arthritis, and in 90% of patients are positive for rheumatoid factor. The characteristic symptoms of this disorder are dry mouth and dry eyes. In addition, sjögren's syndrome can cause dry skin, nose and vagina, and may affect other organs of the body, including the kidneys, blood vessels, lungs, liver, pancreas and brain. Nine out of ten of pale�tov with Sjogren's syndrome are women, and the average age at onset of the disease is close to fifty years, although Sjogren's syndrome occurs in all age groups in both men and women. This disease is set at 4 million people in the U.S. alone, bringing it to second place among the most common autoimmune rheumatic diseases.

Myositis is a common condition characterized by inflammation of skeletal muscles or arbitrarily-twitch muscles. Inflammation of the muscles can be caused by an allergic reaction, exposure to toxic substances or drugs, other disease, such as cancer or rheumatoid conditions, or by a virus or other infectious agent. Chronic inflammatory myopathy are idiomatum, which means the cause is unknown. It is established that they are autoimmune disorders in which the white blood cells (which normally overcome the disease attack the blood vessels, normal muscle fibers and connective tissue in organs, bones and joints.

Polymyositis affects skeletal muscles (involved in movement) on both sides of the body. This disease is rarely observed before the age of 18 years; in most cases, the age of patients ranged from 31 to 60 years. In addition to the above symptoms muscle weakness progresses, to�area leads to difficulty swallowing, speech, rise from a sitting position, climbing stairs, lifting things, raise the hands above the head. In patients with polymyositis also manifested arthritis, shortness of breath and cardiac arrhythmia.

Dermatomyositis is characterized by skin rash that precedes or accompanies progressive muscle weakness. The rash is stained with a color change to blue-red or red and it is characterized by the development on the eyelids and muscles involved in the flexion and extension of joints, including knuckles, elbows, heels, and toes. Red rash may also be found on the face, neck, shoulders, upper chest, back and other places and they can be swollen in the areas of the lesion. Red rash sometimes occurs without obvious involvement of the muscles. Adults with dermatomyositis may occur loss of body weight and weak manifestation of fever, inflamed lungs and sensitivity to light. Dermatomyositis adults unlike polymyositis may accompany tumors of the breast, lung, female genital tumors or bowel. In children and adults with dermatomyositis may occur calcium deposition, which manifests itself in the form of a solid growths under the skin or into a muscle (calcification). In most cases, the calcification occurs every 1-3 years after onset, but may occur many years later. Such deposits are often observed with de�Malaisie in children than dermatomyositis, which develops in adults. Dermatomyositis may be associated with collagen-vascular or autoimmune diseases.

Myositis cells with inclusions (MTV) is a progressive muscle weakness and exhaustion. MTV is similar to polymyositis, but has specific differences. The beginning of muscle weakness generally increases gradually (over months or years) and affects proximal and distal muscles. Muscle weakness may affect only one side of the body. Small holes, called vacuoles, were observed in the cells in the affected muscle fibers. Falling and tripping are usually the first noticeable symptoms of MTV. In some patients, the disorder starts with weakness in the wrists and fingers, which causes the difficulty, if you need something to do up, to fasten or stick. May be weakness of the muscles of the wrist and fingers, and atrophy (thinning or loss of muscle mass) muscles of the forearm and quadriceps muscles of the legs. Difficulty in swallowing patients experience with MTV in about half of cases. The symptoms of this disease usually begin after the age of 50 years, although it can occur earlier. Unlike polymyositis and deatomizer MTV occurs more frequently in men than in women.

Juvenile myositis sometimes resembles dermatomyositis � the adult polymyositis. It usually affects children aged 2-15 years with symptoms that include weakness and inflammation of the proximal muscles, edema (violation of the outflow of fluid in body tissues that causes swelling), muscle pain, fatigue, skin rash, fever, and contractures (chronic contraction of muscles or tendons around the joints caused by inflammation of the tendons around the muscles, which do not allow the joints to move freely). Children with juvenile myositis may also have difficulty swallowing and breathing, and can also be affected heart. Approximately 20-30% of children with juvenile dermatitis develops calcios. In the blood of children with juvenile dermatitis the level of the muscle enzyme creatine kinase may be normal, but levels of other muscle enzymes exceed the norm. Thus, in other embodiments of the present invention the antibodies of the present invention can be used for prevention, treatment or relief of myositis, inflammatory myositis, idiopathic myositis, polymyositis, dermatomyositis, myositis cells with inclusions (MTV), juvenile myositis, or symptoms associated with these conditions.

In another embodiment of the present invention the antibodies of the present invention may be useful for preventing, treating or alleviating the symptoms associated with vasculitis.

�ntitle of the present invention may be useful for the treatment of scleroderma. Treatment scleroderma described in patent applications US 60/996175, published 5 November 2007, and patent application PCT PCT/US2008/82481, the essence of which is included in the present invention in the form of links.

In another embodiment of the present invention the antibodies of the present invention may be useful for prevention, treatment or relief of symptoms associated with sarcoidosis. Sarcoidosis (also called disease Besnier-Beck) is a disorder of the immune system, characterized necroticism granulomas (small nodules of inflammation). Almost may be affected of any body; however, the granuloma is most often manifested in the lungs or lymph nodes. Symptoms can sometimes appear suddenly, but usually appears gradually. X-ray of the lungs sarcoidosis can have the appearance of tuberculosis or lymphoma.

Within the scope of the present invention also provides kits comprising the compositions (e.g., anti-IFNAR1 antibodies) of the present invention and instructions for use. The kit may also include at least one additional reagent, or one or more additional antibodies of the present invention (e.g., an antibody having a complementary activity which binds to an epitope on the antigen target, different from the first antibody). Kits usually�about include the label indicate the appropriate use of the contents of the set. The label given any written or electronically presented information from the supplier invested in a set, or attached to the outside, or otherwise attached to the set.

Combination

The compositions of the present invention may also be administered in combination therapy, for example, combined with other agents. For example, combination therapy can include an anti-IFNAR1 antibody of the present invention in combination with at least one other immunosuppressant.

In some methods, two or more monoclonal antibodies with different binding specificity administered simultaneously, with the dosage of each entered antibody falls within the specified ranges. The antibody is usually administered for a variety of reasons. Intervals between single dosages can be, for example, weeks, months, every three months and a year. Intervals can also be irregular according to the testimony of measuring blood levels of antibody to the antigen target in the patient. In some methods, the dose is adjusted so as to achieve the concentration of antibodies in the plasma of about 1-1000 μg /ml and in some methods about 25-300 μg/ml.

If antibodies to IFNAR1 is administered together with another agent, they may be given in any �Radke or simultaneously. For example, the anti-IFNAR1 antibody of the present invention may be used in combination with one or more of the following agents: medicines containing mesalamine (including sulfasalazine and other agents containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide), nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g., predinisone, hydrocortisone), TNF-inhibitors (including adalimumab (product HUMIRA®), etanercept (product ENBREL®) and infliximab (product REMICADE®)), of immunosuppressants (such as 6-mercaptopurine, azathioprine and cyclosporine a) and antibiotic anti-IFNα antibodies, antibodies to IFNγ receptor, and soluble IFNγ receptor. In addition, the anti-IFNAR1 antibody of the present invention can be used in combination with an antagonist of Flt3 ligand (see, e.g., US patent application 2002/0160974).

In other embodiments of the present invention the compositions of the present invention may also include agents used for the treatment of SLE. Such agents include analgesics, corticosteroids (e.g., predinisone, hydrocortisone), immunosuppressants (such as cyclophosphamide, azathioprine and methotrexate), antimalarial drugs (e.g., hydroxychloroquine), and biological medicines, which inhibit production dndn� antibodies (e.g., LJP 394).

Equivalents

Specialists in this field can set or find out, using only routine experimentation, many equivalents to the embodiments of the present invention contained in the description.

All publications, patents and patent applications mentioned in the present description, included in it by reference in their entity to the same extent as if each individual publication, patent or patent application were specifically and separately shown in the present invention by reference.

In addition, the provisional patent applications US 61/006962, 61/034618 and 61/049970 included in the present invention in the form of links to their essence.

Specific embodiments of the present invention

1. Modified monoclonal antibody of class IgG specific against IFNAR1, wherein the said antibody comprises in the Fc region at least one amino acid substitution selected from the group consisting of L234F, L235E, and P331S, the numbering of which is given according to the EU index, developed by Kabat, wherein the said antibody exhibits reduced affinity for at least one Fc ligand as compared with unmodified antibody.

2. The antibody according to embodiment of the present invention 1, wherein the said antibody belongs to the subclass IgG1 or IgG4.

3. The antibody according to embodiment implemented�I 2 of the present invention, moreover, the specified antibody is an IgG1 molecule class.

4. The antibody according to embodiment of the present invention 3, wherein the said antibody comprises the amino acid substitution P331S.

5. The antibody according to embodiment of the present invention 3, wherein the said antibody comprises the amino acid substitution: L234F and L235E.

6. The antibody according to embodiment of the present invention 3, wherein the said antibody comprises the amino acid substitution: L234F, L235E and P331S.

7. The antibody according to embodiment of the present invention 3, wherein the said antibody is an IgG4 molecule class.

8. The antibody according to embodiment of the present invention 7, wherein the said antibody comprises the amino acid substitution L235E in the field Fc.

9. The antibody according to embodiment of the present invention 7, wherein the said antibody further comprises amino acid substitution S228P in the field Fc.

10. The antibody according to one of embodiments 1-9 of the present invention, wherein the said antibody comprises at least one region, complementarity determining (CDR) selected from the table.2.

11. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. variable region CDR1 of the heavy chain that includes Seq ID NO:31;

b. variabel�ing the CDR2 region of the heavy chain of human comprising Seq ID NO:32;

V. variable region CDR3 of the heavy chain that includes Seq ID NO:33;

G. variable region CDR1 light chain that includes Seq ID NO:34;

D. variable region CDR2 light chain that includes Seq ID NO:35; and

E. variable region CDR3 light chain that includes Seq ID NO:36.

12. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. variable region CDR1 of the heavy chain that includes Seq ID NO:1;

b. variable region CDR2 of the heavy chain that includes Seq ID NO:2;

V. variable region CDR3 of the heavy chain that includes Seq ID NO:3;

G. variable region CDR1 light chain that includes Seq ID NO:4;

D. variable region CDR2 light chain that includes Seq ID NO:5; and

E. variable region CDR3 light chain that includes Seq ID NO:6.

13. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. variable region CDR1 of the heavy chain that includes Seq ID NO:11;

b. variable region CDR2 of the heavy chain that includes Seq ID NO:12;

V. variable region CDR3 of the heavy chain that includes Seq ID NO:13;

G. variable region CDR1 light chain that includes Seq ID NO:14;

D. variable region CDR2 of light C�PI man comprising Seq ID NO:15; and

E. variable region CDR3 light chain that includes Seq ID NO:16.

14. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. variable region CDR1 of the heavy chain that includes Seq ID NO:21;

b. variable region CDR2 of the heavy chain that includes Seq ID NO:22;

V. variable region CDR3 of the heavy chain that includes Seq ID NO:23;

G. variable region CDR1 light chain that includes Seq ID NO:24;

D. variable region CDR2 light chain that includes Seq ID NO:25; and

e. variable region CDR3 light chain that includes Seq ID NO:26.

15. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. the variable region of the heavy chain of a human, comprising the amino acid sequence of Seq ID No:38; and

b. variable region light chain that includes the amino acid sequence of Seq ID No:40.

16. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. the variable region of the heavy chain of a human, comprising the amino acid sequence of Seq ID No:8; and

b. variable region light chain that includes the amino acid sequence of Seq ID No:10.

17. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. the variable region of the heavy chain of a human, comprising the amino acid sequence of Seq ID No:18; and

b. variable region light chain that includes the amino acid sequence of Seq ID No:20.

18. The antibody according to one of embodiments of the present invention 1-10, wherein the said antibody comprises:

and. the variable region of the heavy chain of a human, comprising the amino acid sequence of Seq ID No:28; and

b. variable region light chain that includes the amino acid sequence of Seq ID No:30.

19. The antibody according to one of embodiments of the present invention 1-18, wherein the said antibody comprises the sequence of a constant region light chain Seq ID No:41.

20. The antibody according to one of embodiments of the present invention 1-18, wherein the said antibody comprises a constant region of the heavy chain of the human sequence Seq ID No:42.

21. The antibody according to one of embodiments of the present invention 1-18, wherein the said antibody comprises a constant region light chain having the amino acid sequence of Seq ID No:41 and a constant region of a heavy chain having the amino acid sequence of Seq I No:42.

22. The antibody according to one of embodiments of the present invention 19-21, wherein the said antibody comprises the amino acid sequence of the heavy chain, including allelic variation and indicated allelic variation is at least one or more selected from the group consisting of 214, 221, 356 and 358 on the numbering system according to the EU index.

23. The antibody according to one of the preceding embodiments, wherein the said antibody is selected from the group consisting of: antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies and synthetic antibodies.

24. Isolated nucleic acid comprising a polynucleotide sequence encoding the antibody according to one of the preceding embodiments of the present invention.

25. Nucleic acid according to embodiment of the present invention 24, and the specified nucleic acid is a replicative vector.

26. Nucleic acid according to embodiment of the present invention 25, the given polynucleotide sequence functionally linked to the promoter.

27. A host comprising or transformed with a vector according to embodiment of the present invention 25 or 26.

28. Transgenic mouse comprising a transgene heavy�Oh and light chains of human immunoglobulin, wherein the mouse expresses an antibody according to one of embodiments of the present invention 1-23.

29. The hybridoma obtained from a mouse embodiment of the present invention 28, wherein the hybridoma produces the indicated antibody.

30. Pharmaceutical composition comprising an antibody according to one of embodiments of the present invention 1-23 and a pharmaceutically acceptable excipient.

31. Method of treatment of a condition or disease associated with an immune disorder, comprising administering to a subject in need, an effective amount of the composition according to embodiment of the present invention 30.

32. A method according to embodiment of the present invention 31, wherein the disease is a disease that is mediated by type I interferon.

33. A method according to embodiment of the present invention 32, wherein the said type I interferon is interferon alpha.

34. A method according to embodiment of the present invention 33, wherein the disease mediated by type I interferon, associated with the receptor interferon type I.

35. A method according to embodiment of the present invention 31, wherein the disease or disorder is HIV infection AIDS.

36. A method according to embodiment of the us�Mr sage of the invention 31, moreover, the disease or disorder is systemic lupus erythematosus.

37. A method according to embodiment of the present invention 31, wherein the disease or disorder is Sjogren's syndrome.

38. A method according to embodiment of the present invention 31, wherein the disease or disorder is myositis.

39. A method according to embodiment of the present invention 31, wherein the disease or disorder is an inflammatory myositis.

40. A method according to embodiment of the present invention 31, wherein the disease or disorder is polymyositis.

41. A method according to embodiment of the present invention 31, wherein the disease or disorder is dermatomyositis.

42. A method according to embodiment of the present invention 31, wherein the disease or disorder is myositis-enabled cells.

43. A method according to embodiment of the present invention 31, wherein the disease or disorder is juvenile myositis.

44. A method according to embodiment of the present invention 31, wherein the disease or disorder is idiopathic inflammatory myositis.

45. A method according to embodiment of the present Fig�plants 31, moreover, the disease or disorder is vasculitis.

46. A method according to embodiment of the present invention 31, wherein the disease or disorder is sarcoidosis.

47. A method according to embodiment of the present invention 31, wherein the disease or disorder is selected from the group consisting of: inflammatory bowel disease, multiple sclerosis, autoimmune tiroides, insulin-dependent diabetes mellitus, glomerulonephritis and disease graft-versus-host.

48. A method according to embodiment of the present invention 31, wherein the disease or disorder is psoriasis or a condition caused by psoriasis.

49. A method according to embodiment of the present invention 31, wherein the disease or disorder is graft rejection or disease graft-versus-host.

50. A method according to embodiment of the present invention 31, wherein the disease or disorder is selected from the group including: graves ' disease, Hashimoto's thyroiditis, Crohn's disease, psoriasis, psoriatic arthritis, sympathetic ophthalmic, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome, sjögren syndrome, sclero�RMU, Addison's disease, polyendocrine syndrome of deficiency, Guillain-Barre syndrome, immune thrombocytopenic purple, pernicious anemia, myasthenia gravis, primary biliary cirrhosis, mixed connective tissue disease, vitiligo, autoimmune uveitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, celiac disease, dermatitis herpetiformis, autoimmune hepatitis, pemphigus, pemphigus vulgaris, pemphigus foliaceous, bullous pemphigoid, autoimmune myocarditis, autoimmune vasculitis, alopecia alopecia, autoimmune atherosclerosis, Behcet's disease, autoimmune myelopathy, autoimmune hemophilia, autoimmune interstitial cystitis, autoimmune diabetes insipidus, autoimmune endometriosis, recurrent polyhedric, ankylosing spondylitis, ankylosing spondylitis, autoimmune urticaria, dermatomyositis, syndrome of Miller Fisher, IgA nephropathy, goodpasture's syndrome and herpes pregnant women.

51. Method according to one of embodiments of the present invention 31-50, further comprising administering at least one agent selected from the group consisting of: phototherapy, corticosteroids, prednisone, NSAID, plasmapheresis, immunosuppressants, methotrexate, retinoic acid, Tg, mycofenolate of mofetil, esters Fuma�acid type, cyclophosphamide, azathioprine, cyclosporine, and immunoglobulins.

52. Method according to one of embodiments of the present invention 31 to 51, further comprising administering at least one agent selected from the group consisting of: alefacept (product AMEVIVE™), etanercept (product ENBREL®), adalimumab (product HUMIRA®), infliximab (product REMICADE®), belimumab (product LYMPHOSTATB™), rituximab (product RITUXAN®) and efalizumab (product RAPTIVA®).

53. The crystal comprising the Fc region of human IgG, wherein the Fc region of human IgG comprises at least one amino acid substitution selected from the group consisting of L234F, L235E, and P331S, the numbering of which is given according to the EU index, developed by Kabat, and in which the said fragment exhibits reduced affinity in relation to at least one Fc ligand compared with the unmodified Fc region.

54. The crystal according to embodiment of the present invention 53, wherein the Fc region of human IgG consists of amino acid substitution L234F, L235E and P331S.

55. The crystal according to embodiment of the present invention 53, wherein the diffraction quality.

56. The crystal according to embodiment of the present invention 53, which is the native crystal.

57. The crystal according to embodiment of the present invention 53, characterized rhombic elements�tare cell a=50,18±0,2 Å, b=147,30±0,2 Å and c=75,47±0,2 Å.

58. The crystal according to embodiment of the present invention 53, which has a space group of C2221.

59. Modified monoclonal antibody, wherein the said antibody comprises in said Fc region amino acid substitution L234F, L235E, and P331S, the numbering of which is given according to the EU index, developed by Rabat, and the antibody exhibits reduced affinity in relation to at least one Fc ligand as compared with unmodified antibody.

60. A fusion protein comprising a modified Fc region, wherein the Fc region comprises amino acid substitution L234F, L235E, and P331S, the numbering of which is given according to the EU index, developed by Kabat, and the Fc region exhibits lower affinity in relation to at least one Fc ligand compared to the Fc region.

61. A method of producing the antibody according to one of embodiments of the present invention 1-23 or 59.

62. The antibody according to one of embodiments of the present invention 1-23 or 59, wherein the said antibody is internalizers antibody.

63. A fusion protein according to embodiment of the 60, with specified hybrid protein is internalizers hybrid protein.

64. A fusion protein according to embodiment of the 63, with specified hybrid protein specifically binds � IFNAR1.

65. The antibody according to one of embodiments of the present invention 1-23, 59 or 62, wherein the said antibody exhibits reduced or truncated antibody-dependent cellular cytotoxicity (ADCC) compared with the specified non-modified antibody.

66. The antibody according to one of embodiments of the present invention 1-23, 59 or 62, wherein the said antibody exhibits reduced or truncated complement-dependent cytotoxicity (CDC) compared with the specified non-modified antibody.

67. The antibody according to one of embodiments of the present invention 1-23, 59 or 62, wherein the said antibody exhibits reduced or truncated cytotoxicity ADCC and CDC compared with the specified non-modified antibody.

Sequence

Constant region light chain (SEQ ID No:41)

The constant region of the heavy chain (SEQ ID No:42)

Examples

The present invention is described below with reference to examples. These examples are provided for illustration only and the present invention in any way is not restricted by them, and these examples are provided to cover any or all variations that result in the present invention methods.

Example 1. The IHC profile of many anti-IFNAR1 antibodies

Goal. �to assess the IHC profile of anti-IFNAR1 antibodies on a set of different fabrics.

Methods. Immunohistochemical techniques to study the properties of binding of the antibodies known in the field and, for example, can be accomplished by highlighting the desired cells or tissue and preparing them for microscopic examination using standard fixation and methods of consolidation on the surface.

Macrophages of mice. The cell suspension pelleted by centrifugation for formation of the precipitate. The precipitate was frozen in medium for frozen OST for the formation of the block. Slices cut into layers with a thickness of 5 μm, 10 min soak in acetone and left to dry with desiccant overnight. Before use, sections were immersed in 10% formalin in neutral buffer for 10 sec and washed 3 times in buffer (1× TBS with 0.01% Tween20).

The monocytes. The cell suspension moisten/put a drop directly on glass slides. The slide is left to dry overnight and then soaked in acetone for 10 min and left to air dry. Before applying the glass completely immersed in 10% neutral buffered formalin for 10 h and washed 3× in buffer (1× TBS with 0.01% Tween20).

The tissues of the brain and heart of man. Tissue samples from the donor are frozen in the medium for frozen OST for the formation of the block. Cut into slices with a thickness of 5 μm, soaked in acetone for 10 min and left to dry with pegloticase over night. Before use, sections were immersed in 10% neutral buffered formalin for 10 h and washed 3 times in buffer (1× TBS with 0.01% Tween 20).

Applying the label to the antibody. Antibodies kongugiruut with Biotin according to the following Protocol. Approximately 500 µg of antibody was mixed with Biotin, which are taken in 20-fold excess and incubated for 2 h in the dark at 4°C. After 2 h incubation the mixture of antibody/Biotin contribute to pre-balanced PD10 column with 1X PBS. Subsequently conjugated with Biotin antibody is concentrated to the desired concentration using the vial for concentration YM-30 Centricon.

Staining of sections. After washing in buffer, sections were treated to suppress endogenous peroxidase by treatment with a solution of glucose oxidase (1 U/ml, Sigma G0543), B-D(+) glucose (10 mm, Sigma G5250), sodium azide (1 mm, Sigma 8032) for 1 h at room temperature. Then sections were rinsed with buffer for washing (1× TBS with 0.01% Tween 20), Sections were placed in the solution for blocking protein (1× PBS pH 7.2, 0.5% casein (N-Z amine, Sigma C0626), 1% BSA (Sigma A7906), and 1.5% normal goat serum (Jackson Labs #005-000-001) for 30 min at room temperature. Biotinylated antibody (see above) is applied to the slices by dilution in blocking solution of protein. Incubation of slices with biotinylating the antibody is carried out at room temperature for 2 h. Slices �polaskia 3 times with wash buffer (1× TBS, 0.01% of Tween 20). Detection of antibodies is carried out using the Vectastain kit Kit (firm Vector Laboratories). Sections were washed and again stained using hematoxylin. Sections were dehydrated and covered with coverslip before viewing.

Results. Fig.6A presents the results of the IHC analysis of tissue samples of the human brain, colored by a variety of anti-IFNAR1 and control antibodies. Antibody MDX-1333 (75 μg/ml) and 4G5 (50 μg/ml) showing strong staining of brain tissue, which is expressed in the form of a brown/dark staining observed throughout the sample surface. Antibody 9D4 (50 μg/ml), and MDX-1333 and 4G5, do not stain the tissue sample of the human brain that is shown in the form of low brown/dark staining throughout the sample. Control isotype IgG1 include, to show the specificity of binding of specific antibodies.

Fig.6B presents the results of the IHC analysis of monocytes, painted various anti-IFNAR1 and control antibodies. All antibodies MDX-1333 (50 and 20 µg/ml), 4G5 (50 μg/ml) and 9D4 (50 and 20 µg/ml) show a pronounced staining of monocytes, which manifests itself in the form of a brown/dark staining of the samples. Isotype control antibody R3-47 (50 μg/ml) exhibits a pronounced staining of human monocytes. In addition, MDX-1333 (50 μg/ml) does not stain cleaned macropha�and mouse.

Conclusions. In the IHC study, the anti-IFNAR1 antibody 9D4 exhibits a reduced level of staining than other anti-IFNAR1 antibodies, for example, MDX-1333 and 4G5.

Example 2. Obtaining antibodies 9D4 TM

The modified anti-IFNAR1 antibody, referred to as "9D4-TM" is produced by the following procedure.

The human receptor γ1 Fc cloned and constructed from peripheral blood lymphocytes (PBL) of human primary allocation of total RNA, transcribing cDNA and PCR amplification constant regions with gene-specific primers containing the restriction sites Ara I and EcoRI for cloning into the vector mammals REE. A triple mutant (TM) includes three changes of amino acids in human IgG to reduce ADCC effector function (L234F, L235E and P331S). TM design, using a human IgG1 (KOL) as a matrix and site-directed mutagenesis (QuickChange XL, firm Stratagene) to encode changes of three residues in the Fc region. The sequence of the mutagenic primers for the coding changes L234F/1235E/P331S following:

MD1056=5' cgtgcccagcacctgaaTtcGAggggggaccgtcagtcttc 3'L234F, L235E direct (SEQ ID NO:43)

MD1057=5' gaagactgacggtccccccTCgaAttcaggtgctgggcacg 3' L234F, L235E reverse (SEQ ID NO:44)

MD1058=5' ccaacaaagccctcccagccTccatcgagaaaaccatctcc 3' P331S direct (SEQ ID NO:45)

MD1059=5' ggagatggttttctcgatggAggctgggagggctttgttgg 3' P331S reverse (SEQ ID NO:46)

The clones encoding the antibody 9D4-TM, sequeiros sequence to confirm triple Muta�AI and determine on ABI3100 genetic analyzer.

Example 3. Obtaining antibodies 9D4 DM

The modified anti-IFNAR1 antibody, referred to as "9D4-DM" is produced by the following procedure.

Region γ4 human Fc cloned and design of timber for the primary allocation of total RNA, cDNA transcription and PCR amplification constant regions with gene-specific primers containing the restriction sites Ara I and EcoRI for cloning in the vector mammals REE.

The double mutant (DM) includes two mutations in the Fc region of IgG4 human S228P and L235E. Mutagenic primers for the coding of DM include:

MD1060=5' ggtcccccatgcccaCcatgcccagcacctg 3' hinge S228P direct (SEQ ID NO:47)

MD1061=5' caggtgctgggcatgGtgggcatgggggacc 3' hinge S228P reverse (SEQ ID NO:48)

MD1062=5' ccagcacctgagttcGAggggggaccatcagtc 3' IgG4 L234F, L235E direct (SEQ ID NO:49)

MD1063=5' gactgatggtccccccTCgaactcaggtgctgg 3' IgG4 L234F, L235E reverse (SEQ ID NO:50).

Clones encoding the antibody 9D4-DM, sequeiros to confirm changes in the coding and determine on ABI3100 genetic analyzer.

Example 4. Anti-IFNAR1 antibodies inhibit IFN-mediated STAT phosphorylation.

Goal. To establish the ability of anti-IFNAR1 antibodies 9D4-TM to inhibit IFN-mediated STAT phosphorylation in mononuclear peripheral blood cells (PBMC).

Methods. PBMC from healthy human donors are purified, using a medium LSM (firm MP Biomedical, Solon, Oh). Ciav counted and seeded in an amount of 106cells � the hole for a particular status. The antibodies added in an amount of 10 μg/ml in the appropriate well and incubated at 37°C in an atmosphere of 5% CO2within 10 min After pre-incubation with antibodies, recombinant human IFNα2a (firm PBL Biomedical, Piscataway, NJ) or IFN derived plasmacytic dendrocygna cells (MPC) (see below receive supernatants of IFN type I from MPC) is introduced into appropriate wells at a concentration of 100 or 500 IU/ml for 20 min. the Cells are pelleted by centrifugation at 1200 rpm.min for 5 min and washed with sterile 1× PBS. After additional deposition by centrifugation remove the FSB and lisarow cells using a reagent for extraction of mammalian proteins (firm Pierce, Rockford, Il) with the addition of 300 μl 1× cocktails 1 and 2 of phosphatase inhibitors (Sigma, St. Louis, mo) and 1× protease inhibitor (Roche Biomedical, Nutley, NJ). Lysates were incubated for 10 min on a pie rocking chair for the complete lysis, transferred into test tubes for microcentrifuge and pelleted by centrifugation at 14000 rpm to remove residual cells. The sample buffer NuPAGE (Invitrogen company, carlsbad, CA) and dTT (Sigma, St. Louis, mo) was added to the lysates to a final concentration of 1× and all samples denature lock heat at 100°C for about 10 min, 15 μl of each sample added to NuPage 10% Bis-tris poly�crylamide gel (firm Invitrogen, Carlsbad, CA) in NuPAGE MES SDS rolling buffer, anti-oxidant buffer 1× NuPAGE. Samples is carried out at 180 V for 30 min to separate proteins on the strip. The proteins are then transferred onto nitrocellulose membrane and stain block in the buffer 1× FSB (firm Gibco BRL, carlsbad, CA) containing 5% BSA (Sigma, St. Louis, mo) overnight at 4°C. the Blocking medium is then removed and 0.2 μg/ml anti-STAT1, anti-STAT1 pY701 or 1:1000 dilution of β-active antibodies (company Cell Signaling Technology, Danvers, mA) add to the appropriate spots and incubated over night at 4°C. the Spots were washed three times in 1× TBS with 0.05% Tween20 (Sigma, St. Louis, mo). Anti-rubicelle secondary antibody, anywhereman with horseradish peroxidase and diluted 1:25000, add to stains and incubated for 1 h at room temperature. Spot washed as described above and 3 ml of a 1:1 mixture of reagent Supersignal Pico West (firm Pierce, Rockfor, Il) was added to each spot for 1 min. Spot dry, the excess reagent is removed and the bands visualized using the processor Kodak X-omat 1000A.

Results. Fig.7. presents the results of studies on the activation of STAT, in which the cells are stimulated leukocyte IFN in the presence or in the absence of anti-IFNAR1 antibodies. In the absence of antibodies leukocyte interferon stimulates the phosphorylation �toform 1, 3 and 5 STAT. Incubation of cells with antibody 9D4-TM inhibits phosphorylation, mediated by treatment with leukocyte interferon. The cells treated with isotype control antibody R3-47, do not show inhibition of STAT phosphorylation in response to stimulation of leukocyte interferon.

Conclusion. The results in Fig.7 show that the antibody 9D4-TM is able to inhibit responses to IFNα, for example, induction of STAT phosphorylation in mononuclear cells of peripheral blood.

Example 5. Anti-IFNAR1 antibodies inhibit the transmission signal type I IFN.

Goal. Using purified IFN type I cells from the MPC, use of reporter analysis to determine the ability of anti-IFNAR1 antibodies to block the transfer of signal type I IFN.

Methods. Plasmacyte dendrobiinae cells (MPCs) isolated from whole blood of healthy donors, using a medium for the separation of lymphocytes (firm MP Biomedical, Solon, Oh), followed by positive selection, using a set of CD304 (BDCA-4/Neuropilin-l) MicroBead Kit (firm Milteny Biotec, Auburn CA). Purified MPC then cultured at a density of 1×106cells/ml in medium RPMI 1640 enriched with 10% FTS (firm Gibco BRL) and 6 μg/ml CpGA (company InVivogen, San Diego, CA). The supernatants were collected and clarified after 20 h in culture and type I IFN assess quantitatively, using line stably transfected reporter cells HEK29-ISRE against a standard curve of leukocyte IFN (firm PBL Biomedical, Piscataway, New Jersey).

MPC from three healthy human donors used to obtain supernatants derived from MPC humans interferon type I described above. HEK293 cells (ATSC, Manassas, VA) stably transferout the reporter plasmid pHTS-ISRE (firm Biomyx Technology, San Diego, CA) and maintained among DMEM, enriched with 10% FTS, 1× NEAA and 700 μg/ml G418 (Invitrogen company, carlsbad, CA). Cells seeded in the amount of 80000 cells in the hole in the white/transparent 96-well plates Optilix (VWR, West Chester, PA). The appropriate concentration of antibody (611 - 0,00004 nm) contribute to each well and then make the appropriate concentrations of the supernatants with the interferon type I, obtained from the MPC person. Cells, and IFN antibodies were incubated over night at 37°C in atmosphere of 5% CO2and amplification of protein luciferase appreciate legirovaniem cells reagent for lysis of cells (Cell Culture Lysis reagent) and visualization using the Luciferase Assay System (firm Promega, Madison, Wi). The signal measure in imp./h and IC50 values.

Results. The supernatants of IFN type I cells harvested from the MPC, selected from three individual donors. In luciferase reporter study, the incubation of anti-IFNAR1 antibody inhibits the ability to signal transmission with different concentrations of supernatant IFN type I (Fig.8).

Conclusion. These results for�anywayt, what anti-IFNAR1 antibodies capable of inhibiting mediated type I IFN signal transmission by measuring reporter activity in the study.

Example 6. The modified anti-IFNAR1 antibodies exhibit a binding capacity similar to the capacity of the original unmodified antibody.

Goal. To study the binding of IFNAR1 modified antibody compared to the original unmodified versions. Fig.9 shows the curves of binding affinity for antibodies 9D4, 9D4DM and 9D4TM. Binding constants (Kd) for 9D4, 9D4DM and 9D4TM anti-IFNAR1 antibody to determine binding curves.

Methods. 200000 HEK 293F cells seeded in round-bottomed 96-well plates, using 50 μl of RPMI medium 1640 enriched with 10% FTS. Labelled europium antibody 9D4-TM receive under a contract with PerkinElmer Life and Analytical Sciences. To measure nonspecific signal of europium add 25 ál of 100-fold excess of unlabeled serially diluted anti-IFNAR1 antibody to appropriate wells of 96-hole tablet for 5-10 minutes before making labeled antibodies 9D4-TM. Then add 25 μl conjugated with europium serially diluted antibody to appropriate wells, and the cells and antibodies are gently stirred at room temperature for 1-2 h. After incubation for binding 150 μl of cell medium is added to all wells and the plates center�pageroot at 1200 rpm.min for 5 min at room temperature. Quickly the supernatant was decanted and 250 μl of cell medium is added to all wells. Separation by centrifugation and washing is repeated a total of 3 times. The cells then was resuspended in 100 μl of cell medium. 50 µl resuspending cells transferred into 200 ál of enhancer solution DELPHIA (a PerkinElmer company) in yellow tablets for micrometrology DELPHIA and emission of europium measured on the reader Victor2 Multilabel (a PerkinElmer company). The signals measure in imp./h and the value of KD and Utah get using the software GraphPad Prism 4.

Results. Shown in Fig.9 the data show that the modified antibodies 9D4-TM and 9D4-DM show similar binding affinity to IFNAR1 (9D4=0,06±0,02 nm, 9D4-DM=0,06±0,02 nm, 9D4-TM=0,03±0,01 nm) compared to the original unmodified antibody.

Conclusion. The data show that the modified antibodies have similar binding properties with IFNAR1 with the original unmodified antibody.

Example 7. These studies equilibrium binding to antibody 9D4-TM against sIFNαRI

Goal. To determine equilibrium data for the binding of the antibody 9D4-TM, using a soluble receptor IFNAR1 (sIFNAR1).

Methods. Ligand sIFNAR1 coated pellets UltraLink® Biosupport (firm PIERCE, Rockford, Il) at a concentration of 5 μg/ml and 50 μg/ml in the buffer coating (50 mm sodium carbonate buffer, rn) within t�e 1-2 days at 4°C. Pellets coated then separated (soft rotation with variable intensity) from the unreacted ligand solution and gently shake in blocking buffer (1 ml of 1M Tris, pH 8, containing BSA at a concentration of 10 mg/ml) for about 15 min at room temperature (CT). Then the slurry of pellets again pelleted by centrifugation for removal of the blocking solution and the phase of the block repeat for about 2 h at room temperature, using a fresh aliquot blocking buffer. After the step of freezing the granules with a coating stored at 4°C until use. Before using, the granules with a coating of sINFARl transferred into the vial of pellets was resuspended in 27 ml of instrumental rolling buffer (PBS, RS,4 - a 0.02% NaN3), then attach to the device KinExA 3000.

All the equilibrium binding constant (KD) is prepared on the basis of measurements made on the device KinExA 3000 (firm Sapidyne Instruments, Boise, Idaho). Briefly, 9D4-TM IgG receive at a concentration of 1 PM, 10 PM and 50 PM and are divided into three series of test tubes. This range of IgG concentrations develop for the implementation of measurements and a receptor-, and KD-controlled conditions. Two-fold serial dilutions of ligand sINFAR1 then titrated with the indicated IgG solutions in concentrations ranging from 19.5 FM to 1 nm. According to the supplier's theoretical �curves models the availability based software firm Sapidyne Instruments, Boise, Idaho), and these equilibrium mixtures were incubated for approximately 2-6 hours at room temperature. By the end of this period conducted experiments to test the signal to determine the operating conditions. Detection of free antibodies is possible using species-specific Cy5-labeled secondary antibody reagent (Cy5 AffiniPure F(ab')2 fragment goat anti-human IgG, part No. 109-176-097, firm Jackson Immuno Research Laboratories) used at a concentration of 0.1, 1.0 or 2.0 μg/ml PBS, RS,4 - a 0.02% NaN3containing BSA at a concentration of 1 mg/ml. the experimental data are then jointly choose, using the indicator received via built based software n-curve, to obtain the specified binding constants (KD).

Results. Fig.10A shows the binding curves for three concentrations of 9D4-TM (1 PM, 10 PM and 50 PM) sIFNαRI. Data from at least three independent experiments are selected by obtained by using the software curve to obtain the relative values of KDfor antibody 9D4-TM. The value of KDantibodies 9D4-TM in this analysis, the binding is determined equal to 1.1 PM with 95% confidence interval from 0,603 PM to 1.8 PM. The percentage error in the determination of KD1,1 RM amounted to 1.96%. It was also found that the values of Kon and'koff for antibodies 9D4-TM be 7×106 Ą 1.3×106h-1and 7.7×10-6±1,57×10-61/msec, respectively (data not shown).

Conclusion. The modified anti-IFNAR1 antibody 9D4-TM shows a very low value of KDabout component of 1.1 PM for sIFNAR1 by definition KinExa method.

Example 8. Determination of the affinity of the antibody 9D4-TM on human PBMC

Goal. Determination of binding affinity with human PBMC.

Methods. Mononuclear cells of peripheral blood from healthy human donors are purified, using a medium LSM (firm MP Biomedical, Solon, Oh). Cell count and 200000 cells seeded in round-bottomed 96-well plates, using 50 μl of RPMI medium 1640 enriched with 10% FTS. Labelled europium antibody 9D4-TM get under contract with firms PerkinElmer Life and Analytical Sciences. To measure nonspecific signal of europium add 25 ál of 100-fold excess unlabeled serially diluted 9D4-TM in appropriate wells of 96-hole tablet for 5-10 min before adding the labeled 9D4-TM. 25 μl conjugated with europium serially diluted 9D4-TM was then added in some of the wells and the cells with antibodies gently stirred at room temperature for 1-2 h. After incubation for binding 150 μl of cell environment contribute to the wells and the plates are precipitated by centrifugation at 1200 rpm.min for 5 min at room temperature. Over�Sadok quickly decanted and 250 μl of cell environment contribute to all wells. Separation by centrifugation and washing is repeated a total of three times. Then the cells was resuspended in 100 μl of cell medium. 50 µl resuspending cells transferred into 200 µl DELPHIA amplifying solution (a PerkinElmer company) in yellow tablets for micrometrology DELPHIA, and the emission of europium measured on the reader Victor2 Multilabel (a PerkinElmer company). The signals measure in imp./h and Kd values In max gain, using the software GraphPad Prism 4.

Results. Using the measurements of affinity, are shown in Fig.10B, determines that the Kd of binding of 9D4-TM with ciav person is 0.29 nm ± 0,11 nm when the number of binding sites 1448±447. Using similar approaches found that the constant binding affinity for IFNAR of macaques graboid 0.65±0,42 nm when the number of binding sites 648±204 (data not shown).

Conclusion. The results presented in Fig.10B show that the antibody 9D4-TM specifically binds with a high affinity with human PBMC.

Example 9. The modified anti-IFNAR1 antibodies show a similar effect with the original unmodified antibody.

Goal. Show that the modified anti-IFNAR2 antibodies (i.e., anti-IFNAR1 antibodies with reduced affinity to Fc ligand) show a similar effect with the original unmodified antibodies.

Methods. The luciferase system �Porterage research used in the example described above (see example 3). Antibodies to IFNAR1 used in this example are 9D4, 9D4-DM and 9D4-TM, MDX-1333. As a control antibody R3-47.

Results. Using luciferase reporter system receive IC50 values for various anti-IFNAR1 antibodies described above (see Fig.11A). Anti-IFNAR1 antibody 9D4 (0.01 nm) and modified antibodies, for example 9D4-DM (0,01 ppm) and 9D4-TM (0,02 nm), each give rise to a similar IC50 value in the reporter survey, showing that they exhibit similar activity. Another anti-IFNAR1 antibody, MDX1333 (0,04 nm), also exhibits similar activity with unmodified antibody 9D4. Isotype control did not inhibit mediated type I IFN signal transmission in such a luciferase reporter study.

Conclusion. The modified anti-IFNAR1 antibodies show a similar effect with unmodified versions that can be seen from the IC50 values obtained in the luciferase reporter system research, designed for counting cases, IFN signal transduction.

Example 10. 9D4-TM inhibits the action of numerous isoforms of alpha interferon type I

Goal. To show that 9D4-TM inhibits signal transmission characteristic of the specific and numerous isoforms of interferon alpha.

Methods. The luciferase reporter systems research, used�traveler in the present example, was described above (see example 5).

Results. IC50 values for indirect antibody 9D4-TM inhibition of interferon type I are presented in table.4.

Table 4.
IC50 values for indirect 9D4-TM inhibition of interferon type I
Interferon type I9D4-TM IC50 (nm)
IFN-α2b0,07±0,01
IFN-α2aOf 0.3±0.2
IFN-α60,04±0,01
IFN-α160,02±0,03
IFN-α80,03±0,04
IFN-α100,01±0,01
Leukocyte interferon0,01±0,01
IFN-α170,04±0,03
IFN-α140,02±0,01
IFN-α10,004±0,01
IFN-α210,01±0,002
IFN-α7 cameras 0,04±0,01
IFN-α4b0,02±0,01
IFN-β16,8±9,4
IFN-ω0,1±0

It is shown that 9D4-TM exhibits an IC50 value in subnanomolar range for numerous isoforms of interferon alpha leukocyte interferon and interferon omega.

Conclusion. The modified anti-IFNAR1 antibody 9D4-TM exhibits the ability to inhibit signal transmission due to the variety of specific subtypes of interferon-alpha and leukocyte interferon alpha in the reporter research.

Example 11. Determination of the isoelectric points of antibodies 9D4, 9D4DM and 9D4TM

Goal. To assess the biophysical properties of the original unmodified antibody 9D4 and compare with the modified antibodies 9D4-DM and 9D4-TM.

Methods. Analysis of native isoelectric focusing in polyacrylamide gelelectrophoresis (IEF-PAGE) is carried out as follows. In ready ampholine gels (firm Amersham Biosciences, pI range 3.5 to 9.5) contribute 8 µg of protein. The protein samples deleteroute in 10 mm histidine pH 6 before loading on the gel. A wide range of standards pI markers (firm Amersham, pI range 3-10, 8 μl) is used to determine the relative value of pI for Mab. Electrophoresis was performed at 1500 V, 50 mA in t�within 105 min. The gel is fixed for 45 min, using the fixing solution Sigma (5×), diluted with purified water to 1×. Stained overnight at room temperature, using a simple blue dye (firm Invitrogen). Removal of dye is carried out in a solution containing 25% ethanol, 8% acetic acid and 67% of clean water. The isoelectric point determined using a densitometer Bio-Rad GS-800 with the software Quantity One Imaging Software.

Results. Fig.12A presents the definition of the isoelectric point (isoelectric point - pI) for antibodies 9D4WT, 9D4DM and 9D4TM. Samples of the antibodies was investigated by the methods described above, showing the following distinctive features. Antibody 9D4 WT exhibits an expressed protein band, corresponding to 8.2, to 8.35 and 8,51. Antibody 9D4 DM shows one of the expressed protein band corresponding to 7.13. Antibody 9D4 TM exhibits an expressed protein band corresponding to 8,09 and 8,18.

Conclusion. According to the presented in the example, the modified antibodies 9D4-DM and 9D4-TM show a very similar biophysical properties (pI) with the original unmodified antibody 9D4.

Example 12. Thermostability 9D4, 9D4-DM and 9D4-TM

Goal. To assess the biophysical characteristics of the original unmodified antibody 9D4 compared with the modified antibodies 9D4-DM and 9D4-TM.

Methods. Differential scanning calorimetry is maintained�t as follows: melting point (T m) is measured using a VP-DSC (MicroCal company, LLC) using a scan rate of 1.0°C/min and the temperature range 20-110°C. the filtration Period of 8 h is used along with a preliminary scan for 15 min, the Samples obtained by dialysis into 10 mm histidine-HCl, pH 6, using dialysis cassettes from Pierce (3,5 kDa). The concentration of Mab amounts to 0.14 mg/ml, 0.79 mg/ml and 0.64 mg/ml, by definition, when A280. The melting point is determined by following the manufacturer's recommendations and using the software Origin, supplied with the system. In short, heterogeneous baseline move with the buffer and in the sample and in the control cells, to establish thermal equilibrium. After subtracting the baseline data from thermograms of the sample, normalize the data by concentration.

Results. Antibodies 9D4, 9D4-DM and 9D4-TM was subjected to differential scanning calorimetry, have been described above (see results in Fig.12B). Each of the investigated antibodies showing similar melting temperature in the present study. Specifically, the antibodies exhibit the following melting point: 9D4 WT=70,41°C, 9D4-DM=70,41°C, and 9D4-TM=70,88°C.

Conclusion. According to the presented in the example, the modified antibodies 9D4-DM and 9D4-TM show a very similar biophysical properties (Tmwith the original unmodified antibody 9D4.

Example 13. Surrogates anti-FNAR antibodies protect mice from induced IFNα proteinuria

Goal. To show that anti-IFNAR antibodies protect mice from induced proteinuria in models of SLE.

Methods. Female mice NZB/W F1 receive from the firm's Jackson Labs and kept in conditions that prevent ingress of pathogens. Recombinant adenoviral vector containing the cDNA of IFNα subtype 5 mice under the control of the promoter/CMV enhancer (Adv-mIFNα5), used for the induction of onset of lupus in these mice. Mice (8 mice per group) aged 8-11 weeks administered a single intravenous injection of 0.3×1010viral particles Adv-mIFNα5. Control animals injected with the same number of control viral particles Adv. In some experiments, the mice administered the injection consistently dose of Adv-mIFNα5 ranged from 0.01×1010to 1,h10viral particles per mouse. To determine the effectiveness of anti-IFNAR1 mice injected in the next 5 days antibody intraperitoneally in an amount of 10 mg/kg, starting from the time of delivery of the virus Adv. When proteinuria urine testing, using measuring rod (Chemstrip 2 GP; Roche Diagnostics). Proteinuria is estimated at 1 point for levels 30 mg/DL, 2 to 100 mg/DL and 3 for levels ≥ 500 mg/DL. Consider that in mice proteinuria, if two consecutive urine specimens assign a rating of "2" or above.

Results. Results adenovirus infected mice treated with anti-IFNAR1 antibodies shown in Fig.13. I m�Shea, infected with Adv-mIFNα5, the manifestation of proteinuria starts after about 3 weeks. Infected mice treated with control IgG antibody mouse is not protected from the beginning of the development of proteinuria during the course of the experiment that is shown in the beginning of the manifestation of proteinuria, which starts after about 4 weeks. In mice which were injected with anti-IFNAR antibodies, no signs of proteinuria during 8 weeks of the study. In mice which were injected with the control adenovirus, no signs of proteinuria throughout the experiment.

Conclusion. Considered together, these data in this example show that the presence of anti-IFNAR antibodies is induced protection against adv-IFN proteinuria in a mouse model in vivo.

Example 14. Anti-IFNAR antibodies block the gene regulation induced IFN type I

Goal. To show that the anti-IFNAR1 antibodies inhibit or reduce gene regulation of interferon type I in the model of SLE in mice.

Methods. From mice used in the experiments in example 13, also take samples for analysis in the present example. RNA is isolated from tissue using a lysing buffer RLT (Qiagen company). For hard tissues (kidney, spleen, skin) every time is used for processing RNA not more than 50 mg tissue. The sample is placed in lysing buffer and lysing matrix A (firm Qbiogene) and treated for 30�K at 4.5 m/sec, using a homogenizer Fastprep24 (company Thermo Electron Corporation, Waltham, mA). To obtain PBMC samples whole blood was centrifuged and the precipitate lisarow in buffer RLT. After lysis the samples are quickly frozen at -80°C until further use. For RNA extraction thawed lysates tissue is first treated using spin Qiashredder column, then equal volumes of 70% ethanol was added to the tissue lysates and RNA purified using mini-kits for spin columns (Rneasy mini spin column, no manufacturer's recommendations.

cDNA prepared from 3 μg of RNA using reverse transcriptase Superscript III primer and oligo-d(T) according to the manufacturer's Protocol (firm Invitrogen, Corp. Carlsbad, CA). Samples of cDNA diluted in water, not containing nucleases, and stored at -80°C.

The expression levels of selected genes measured by PCR real-time TaqMan® using the ABI 7900HT Fast Real-time PCR system (firm Applied Biosystems, foster city, CA). Gene the household of β-actin used for endogenous control. The final volume of reaction mixtures is 20 ál and 1 ál cDNA, 2 ál 20x primers and probes (firm TaqMan® Gene Expression Assays, Applied Biosystems) and 18 ml of the diluted product mixture TaqMan® Fast Universal PCR Master Mix. The conditions of amplification as follows: 20 seconds at 95°C, 50 cycles of 1 sec at 95°C and 20 sec at 60°C. the magnitude of the PT range 0 to 50, with the latter value�and means the absence of product formation. Quantitative assessment of gene expression is carried out using the CT comparative method (Sequence Detector User Bulletin 2; firm Applied Biosystems) and expressed as the ratio of the differences from gene "household".

Results. Interferon type I, congenital expressed in mice (see example 13) leads to induction of several genes. Fig.14 shows a multiple of six changes of genes responsible for type I interferon, in different populations of mice used in this experiment. Especially genes IFIT1, IFI44, IFI202b, CXCL9, CXCL10 and CXCL11 all induced in mice, congenital expressing IFNα and treated with nonspecific mouse IgG. Mouse, congenital expressing IFNα and treated with anti-IFNAR antibodies do not show any induction of the six genes responsible for type I interferon. As a control to demonstrate the specificity encoded by adenovirus IFNα mice were treated with the FSB or the control adenovirus, and they do not show any induction of these 6 genes. These results show that the introduction of anti-IFNAR antibodies can block the induction of the gene responsible for IFN-alpha in vivo in mouse model.

Conclusion. Anti-IFNAR antibodies can block the regulation of genes responsible for type I interferon in mice models of SLE.

Example 15. Anti-IFNAR antibodies block the production of anti-dhnk and anti-SSA/Ro (anti-nuclear antigen) antibodies induced interface�one type I

Goal. To show the ability of anti-IFNAR antibodies to block the production of anti-nuclear antibodies, for example anti-dhnk and anti-SSa/Ro-induced type I interferon in mice models of SLE.

Methods. Preparation and processing of mice shown in example 13. Levels of serum anti-dndn autoantibodies evaluated by ELISA method. Briefly, tablets ELISA, pre-treated poly(L-lysine) (100 µg/ml), cover with activated DNA from calf thymus (5 µg/ml in carbonate-bicarbonate buffer (SIGMA). After incubation over night at 4°C the plates block the FSB/10% FTS. Serum (dilution 1/200) was incubated for 30 min at room temperature. Bound IgG detected using conjugated with peroxidase goat anti-mouse IgG (1/4000) (KPL), added to the plates for 30 min. Binding determined by addition of TMB substrate (KPL) and stop solution (KPL), and the optical density read at 450 nm. Mouse anti-dndn IgG standard serum examined in serial dilution (from 625 ng/ml) (firm Alfa Diagnostic) in each tablet for standardization. Levels of serum anti-SSA/Ro autoantibodies measured by ELISA method (firm Alfa Diagnostic), following the manufacturer's instructions.

Results. Interferon type I congenital protein is expressed in mice (see example 13), which leads to the accumulation of anti-dhnk and anti-SSA/Ro antibodies. Fig.15 performance�aulani relative amount of anti-dhnk (A) and anti-SSA/Ro (B) antibodies in different populations of mice (control adenovirus, Adv-IFNα+FSB, Adv-IFNα+MuIgG and Adv-IFNα+anti-IFNAR) on the measurement by the method of ELISA. Mice infected with a control adenovirus, show a slight accumulation of anti-dndn or anti-SSA/Ro antibodies in this experiment. Mice infected with adenovirus encoding IFNα, and processed FSB, accumulate anti-dhnk and anti-SSA/Ro antibodies. Adv-IFNα infected mice treated with anti-IFNAR antibodies are anti-dhnk and anti-SSA/Ro antibodies than Adv-IFNa-infected mice treated with nonspecific IgG. These results show that treatment with anti-IFNAR antibodies inhibits the accumulation of anti-dhnk and anti-SSA/Ro antibodies in response to congenital expressed type I IFN.

Example 16. Anti-IFNAR antibodies block the production of IP-10 and IL-18-induced interferon type I

Goal. To show the ability of anti-IFNAR antibodies block the accumulation of cytokines induced by IFNα in mice models of SLE.

Methods. From mice used in the experiments in example 13, also take samples for analysis in the present example. The levels of cytokines in serum measured by ELISA method (R&D systems), following the manufacturer's instructions.

Results. Interferon type I, congenital expressed in mice (see example 13), leads to the accumulation of cytokines IP-10 and IL-18. Fig.16 presents the relative number of IP-10 (A) and IL-18 (B) in different populations of mice (FSB, controlselection, Adv-IFNα+MuIgG and Adv-IFNα+Anti-IFNAR), measured by ELISA. Interferon type I, congenital expressed in mice (see example 12), leads to the accumulation of cytokines, IP-10 and IL-18. Mice infected with a control adenovirus, show a slight accumulation of cytokines IP-10 (A) or IL-18 (B) in this experiment. Mice infected with Adv-IFNα and treated with anti-IFNAR antibodies, accumulates less cytokines IP-10 and IL-18 than mice infected with Adv-IFNα and treated with nonspecific IgG. These results show that treatment with anti-IFNAR antibodies inhibits the accumulation of cytokines IP-10 and IL-18 in response to innate murine type I IFN.

Conclusion. Anti-IFNAR antibodies able to block the accumulation of tikokino induced by IFNα in mice models of SLE.

Example 17. Anti-IFNAR antibodies block the production ANA (antinuclear antibodies) - induced type I interferon

Goal. To show the ability of anti-IFNAR antibodies block the accumulation of induced IFNα anti-nuclear antibodies in mice models of SLE.

Methods. From mice used in the experiments in example 13, also take samples for analysis in the present example. Levels of antinuclear antibodies (ANA) are measured using a test kit (ANA Antibody firm Incorporated) with Hep-2 stable substrate and mitotic figures, following the manufacturer's instructions. Serum was serially diluted and incubated with the cells N�R-2 slices and associated anti-nuclear antibody detected using labeled Hi-FITC goat anti-mouse IgG (H+L) (Antibody firm Incorporated). The ANA titer is expressed as the dilution of serum at which ANA is no longer detected.

Results. Congenital expressed in mice interferon type I (see example 13) leads to the accumulation of anti-ANA antibodies. Fig.17 shows the titer of serum anti-ANA antibodies in different populations of mice (without virus, control adenovirus, Adv-IFNα+FSB, Adv-IFNα+MuIgG and Adv-IFNα+Anti-IFNAR) according to measurements by the method of serial dilution staining on cells NER. In mice infected with a control adenovirus, shows a small accumulation of anti-ANA antibodies in this experiment. Mice infected with adenovirus encoding IFNα, and processed FSB, accumulate anti-ANA antibodies. In mice infected with Adv-IFNα and treated with anti-IFNAR antibodies, appears less anti-ANA antibodies than mice infected with Adv-IFNα and treated with nonspecific IgG. These results show that treatment with anti-IFNAR antibodies inhibits the accumulation of anti-ANA antibodies in response to congenital expressed type I IFN.

Conclusion. Anti-IFNAR antibodies able to block the accumulation of anti-nuclear antibodies induced by IFNα in mice models of SLE.

Example 18. Suppression of antibody-mediated plasma of patients with SLE development dendrocygna cells

Goal. The plasma of SLE patients induces the development dendrocygna cells from normal human monocytes. In this� example investigates the purified monoclonal antibody 9D4-TM on the ability to suppress the development dendrocygna cells which is estimated by the ability of antibodies to inhibit the induction of markers CD38 and CD123 on the surface of cells under the action of the plasma of SLE patients.

Methods. Methods were previously described in the patent application US 20006/0029601, the essence of which is included in the present invention by reference. Essentially, the experiments carried out as follows. 25 ml reagent buffy coat was diluted 4 times in phosphate saline buffer (FSB). The sample is separated in a conical test tubes 4×50 ml, and 15 ml of medium for the separation of lymphocytes (firm ICN Biomedicals) layer bottom layer. After 30 min centrifugation at 500 g, the buffy layer of cells containing the mononuclear cells of peripheral blood (PBMC), were recovered and washed with PBS. Cells was resuspended in culture medium containing 1% heat inactivated human serum, to a concentration of 4×106cells/ml Monocytes isolated by incubating PBMC (2,0×107cells/5 ml/25 cm2 flask) for 1.5 h at 37°C in culture medium and then washed twice unattached cells. For the induction of maturation of monocytes cells were incubated with medium containing 25% human plasma from healthy volunteers or from patients with SLE. Research on blocking antibodies is carried out by adding 30 μg/ml of anti-IFNAR1 antibody or isotype control, IgG1, to culture. Cells were incubated for 4 days, washed FSB � treated with a reagent Versene 1:5000 for 10 min at 37°C. If necessary, the cells are separated neat soft scraping before washing and analysis. Each culture was resuspended in staining medium (balanced salt solution Hanks's Balanced Salt Solution with 0.2% sodium bicarbonate, and 0.01% sodium azide, 0.1 mm EDTA, 20 mm HEPES and 2% fetal calf serum) and are uniformly distributed in six wells in 96-well plates with V-shaped bottom. The cells are pelleted by centrifugation with a varying duty Preah 2100 rpm.mines on the rotor Sorvall RTH-750 and was resuspended in 25 μl of staining medium. One microgram specific conjugated with phycoerythrin antibodies contribute to each well and incubated on ice for 45 min, Cells are washed three times, was resuspended in 200 μl 2% paraformaldehyde in PBS and analyzed by liquid cytometry on a flow cytometer Becton Dickinson FACScalibur. Sorting window indicate on the front side against the side of the point of the chart to remove contaminant cells from the analysis.

Results. In this experiment the differentiation of monocytes in dendrobiinae cells in response to IFN, obtained from the plasma of patients with SLE, blocked by treatment with antibody 9D4-TM, measured by expression on the cell surface of the two markers dendrocygna cells, CD38 and CD123. Fig.18. various patient serum samples of patients with SLE are not able to increase the expression of at �poverhnosti cells CD38 and CD123 in the presence of 9D4-TM. The IC50 values of the antibodies 9D4-TM varies from 0.02 nm to 0.06 nm and for CD38, and CD123.

Conclusion. Anti-IFNAR1 antibody 9D4-TM can block the ability of IFNα derived from patients with SLE, to induce the maturation of MPC as measured by the expression of markers on the cell surface.

Example 19. Anti-IFNAR antibodies inhibit the expression of CD38, CD123 and CD86 in monocytes, stimulated leukocyte IFN.

Goal. In this example, antibodies 9D4, 9D4-DM and 9D4 TM explore to suppress the development dendrocygna cells, which assess the ability of antibodies to inhibit the induction of markers CD38 and CD123 on the cell surface of leukocyte IFN.

Methods. Monocytes isolated from whole blood of healthy donors, using a medium for the separation of lymphocytes (firm MP Biomedical, Solon, Oh), followed by positive selection using a kit for isolation of monocytes Monocyte Isolation kit II (firm Milteny Biotec, Auburn, CA). Then purified monocytes were cultured at a density of 1×106cells/ml in medium RPMI 1640 with the addition of 10% FTS (firm Gibco BRL). Serial dilutions of antibodies to get final concentration of 3 μg/ml - 20 ng/ml in the environment and contribute to the appropriate wells with cells. After pre-incubation for about 5 min add 100 IU/ml leukocyte IFN (firm PBL Biomedical, Piscataway, NJ) in certain wells and cultures were incubated at 37°C in atmosphere�Ferre 5% CO 2within 48 hours, then evaluate the expression of CD38 and CD123 on the surface of cells. Briefly, cells pelleted by centrifugation at 1200 rpm.min for 5 min and the culture medium was removed from the monolayers by suction followed by a single washing with sterile PBS. The FSB removed and 1 ml of sterile buffer for dissociation of cells (firm Gibco BRL, carlsbad, CA) or 0.05% trypsin (Invitrogen company, carlsbad, CA) was added into the wells to remove cells from the monolayers. After 5 min, and brief mixing, add equal volumes of RPMI medium 1640 plus 10% of the FST to each well, followed by two series of centrifugation and rinsing with sterile PBS. 50 μl 1× PBS, enriched with 5% BSA (Sigma, St. Louis, Montana) and 10 μg/ml of whole human IgG (firm Jackson ImmunoResearch Laboratories, West Grove, PA) was added to each well to block nonspecific binding of antibodies and Fc were incubated for 10 min at room temperature. Add in appropriate wells 50 μl 1× PBS, enriched with 5% BSA and PE-anti-human CD123 and FITC-anti-CD38 human antibodies (firm Becton Dickinson, Franklin Lakes, NJ)and incubated for 30 min on ice. Cells are washed once in 1× PBS, enriched with 5% BSA, and measure protein expression on the cell surface of the device BD LSRII (firm Becton Dickinson, Franklin Lakes, NJ).

Results. Fig.19 showing�s curves of the inhibition of CD38 expression (A), CD123 (B) and CD86 (B) under the influence of PBMC, stimulated leukocyte IFN, which were incubated with anti-IFNAR antibodies 9D4, 9D4DM and 9D4TM. For every molecule of CD anti-IFNAR antibodies cause similar suppression curves, which are used to obtain IC50 values. For CD38 expression on PBMC, stimulated leukocyte IFN (A), anti-IFNAR antibodies cause the following IC50 values: 9D4=4,3 ng/ml, 9D4DM=40 ng/ml, 9D4TM=25 ng/ml For CD123 expression on PBMC, stimulated leukocyte IFN (B), anti-IFNAR antibodies cause the following IC50 values: 9D4=7 ng/ml, 9D4DM=21 ng/ml, 9D4TM=10 ng/ml For CD86 expression on PBMC, stimulated leukocyte IFN (), anti-IFNAR antibodies cause the following IC50 values: 9D4=20 ng/ml, 9D4DM=20 ng/ml, 9D4TM=26 ng/ml.

Conclusion. The results in this example demonstrate that the antibodies of the present invention 9D4-DM and 9D4-TM show similar curves induced suppression of IFN markers on the cell surface of MPC compared to the original antibody 9D4.

Example 20. The modified anti-IFNAR1 antibodies exhibit reduced binding to the Fc receptor FcγRI.

Goal. Show low specific binding of the Fc receptor with modified anti-IFNAR1 antibodies.

Methods. Action by binding of the modified antibodies 9D4-DM and 9D4-TM with FcγRI (CD64) person evaluated by ELISA method. FcγRI in PBS (pH 7.4) is applied to the wells in amounts of 25 µg/well in a tablet for microtiter�vany (catalog number Costar 3690) at a concentration of 20 μg/ml over night at 4°C. After washing and blocking with 4% milk for 1 h at room temperature, biotinylated 9D4, 9D4TM, 9D4DM and control antibodies are added to the previously blocked tablet and incubated at 37°C for 1 h, starting with a concentration of 500 mg/ml and subsequent twofold serial dilutions. The tablet was washed with the PBS (pH 7.4); containing 0.05% of Tween 20 and added to each well 25 μl HRP conjugated the avidin. After incubation for one hour at 37°C the plates were washed again and add 50 ál/well of substrate SureBlue TMB peroxidase (catalog number KPL 52-00-03). The reaction is stopped with 50 µl 0.2 M H2SO4after 5-10 min. the ELISA Signal is read at 450 nm.

Results. In the analysis of binding based on the ELISA (Fig.20), the modified anti-IFNAR1 antibodies 9D4DM and 9D4TM exhibit reduced binding affinity to FcγRI compared to unmodified 9D4WT antibody and control antibody.

Conclusion. These results show that the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM cause reduced affinity to the Fc receptor FcγRI as compared with unmodified antibody 9D4. Low affinity with the receptor FcγRI may lead to reduced induction of ADCC.

Example 21. Fc receptor FcγRIIIA exhibits reduced binding to the modified anti-IFNAR1 antibodies.

Goal. Show low with�Azania specific Fc receptor with modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM compared with non-modified anti-IFNAR1 antibody 9D4.

Methods. Fifty mg/ml of antibodies 9D4, 9D4TM and 9D4DM, diluted in PBS, applied to the tablet to micrometrology Immunlon IV overnight at 4°C. After washing and blocking with 4% milk for 1 h at room temperature FcγRIIIA variants 158F (low affinity) and 158V (high affinity) with the target Flag is added to the wells blocked tablet, starting with 50 mg/ml, then two-fold serial dilutions. The plates were washed after 1 h and incubated with Biotin conjugated with anti-Flag antibody (Sigma) at a concentration of 2 mg/ml. After washing, 25 µl of HRP conjugated the avidin is added to each well. Unbound materials are removed by washing for 1 h after incubation. The binding signal is detected by TMB substrate.

Results. The results of the research-based binding method between ELISA anti-IFNAR1 antibodies (9D4WT, 9D4DM and 9D4TM) and Fc receptors FcγRIIIA high and low affinity shown in Fig.21(A, B, C). Fig.21(A) 9D4WT antibodies deposited on the tablet ELISA, effectively bind FcγRIIIA receptor with high affinity at concentrations greater than 3 ng/ml, although limited to the binding of FcγRIIIA receptor with low affinity for all studied concentrations. Fig.21(B) modified 9D4DM antibodies deposited on the tablet ELISA, not effectively bind FcγRIIIA receptors with high or low affinity in any of the studied concentrations. Under�cost-effective manner in Fig.21(B) modified 9D4TM antibodies, applied to the tablet ELISA, not effectively bind FcγRIIIA receptors with high or low affinity in any of the studied concentrations.

Conclusion. These results indicate that the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM have very low affinity to FcγRIIIA receptor as compared with non-modified anti-IFNAR1 antibody 9D4. In addition, a low affinity with a specific Fc receptor may lead to reduced ADCC effector function.

Example 22. The modified antibodies 9D4DM and 9D4TM exhibit reduced binding to the Fc receptor FcγRIIIA.

Goal. Show low specific binding of Fc receptor antibodies with modified 9D4DM and 9D4TM.

Methods. Fifty mg/ml of variants of FcγRIIIA (FcγRIIIA-10 158F and FcγRIIIA-10 158V) in PBS was applied to the tablet to micrometrology Immunlon IV over night at 4°C. After washing and blocking with 4% milk for 1 h at room temperature, biotinylated antibodies 9D4, 9D4TM and 9D4DM added to the wells blocked tablet at a concentration of 100 mg/ml. the plate is then washed for 1 h and incubated with HRP conjugated Avidya. Unbound materials are removed by washing for 1 h after incubation. The signal of the binding is determined by TMB substrate.

Results. The results of the analysis of the binding based on the method of ELISA, Fc receptors FcγRIIIA high and low affinity and anti-IFAR1 antibodies (9D4WT, 9D4DM and 9D4TM) shown in Fig.22(A, B, C). Fig.22(A) modified anti-IFNAR1 antibody 9D4 in concentrations greater than 3 ng/ml effectively communicates with the FcγRIIIA receptor with high affinity, immobilized on the tablet ELISA, the antibody shows limited binding to immobilized receptor FcγRIIIA low affinity with all the studied concentrations. Fig.22(B) the modified anti-IFNAR1 antibody 9D4DM not bind effectively immobilized receptors FcγRIIIA with high or low affinity for any of the studied concentrations compared to the unmodified 9D4WT anti-IFNAR1 antibody. In a similar manner in Fig.22(B) the modified anti-IFNAR1 antibody 9D4TM not bind effectively immobilized FcγRIIIA receptors of high or low affinity in any of the tested concentrations compared to the unmodified 9D4WT anti-IFNAR1 antibody.

Conclusion. This example shows that the modified antibodies 9D4DM and 9D4TM exhibit reduced affinity to Fc receptor, FcγRIIIA compared with the parental unmodified antibody 9D4. This low affinity may lead to reduced FcγRIIIA mediated ADCC effector function compared to the parent antibody.

Example 23. Neutralization of IFNα subtypes of anti-IFNAR1 antibodies.

Goal. To show the ability of anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutraliz�tivities of specific subtypes of IFNα reporter in the study.

Methods. Reporter studies on the neutralization of IFNα well documented in this area. In this example, the neutralization of IFNα measured reporter a study based on H1L3. The following is an example of a study of neutralization of IFNα, using cells HiL3 as a reporter. The cells of the human hepatoma line HiL3 transferout a plasmid containing stimulated IFNα response element - luciferase (ISRE-Luc), and the gene of resistance to neomycin. These cells were kindly provided by Dr. Michael Tovey (firm CNRS, Paris, France). Cells Hil3 in the amount of 30,000 cells/well are cultured in a white reflecting 96-well plates (firm DYNEX Microlite) over night in medium Needle in the modification of Dulbecco containing 10% fetal calf serum and 1 mg/ml G418 (+penicillin/streptomycin/L-glutamine). After incubation add various forms of interferon and the plates were incubated for 18 h. the Reaction is done by adding 10 ml of a lytic buffer in a flask with a substrate for luciferase (Luc set Lite Plus kit, the firm Perkin-Elmer); 100 μl of this solution add substrate to each well and read on the counter Top Count for 10 min (10 min of waiting in the dark and reading the wells for 1 h). For each concentration of IFN determine the number of impulses per second (imp./h) and the concentration of IFN or imp./h in each sample are counted at the titration curve IFN, IP�alsoa software Prism software (San Diego, CA) with the parameters of the linear regression.

Results. Neutralizing ability of anti-IFNAR1 antibodies against IFN in HiL3 reporter analysis shown in Fig.23(A-D). Anti-IFNAR1 antibody MDX-1333, 9D4WT and 9D4TM inhibit many subtypes of interferon type I with similar intensity. Anti-IFNAR1 antibody MDX-1333, 9D4WT and 9D4TM neutralize IFNα10 (A) with IC50 values 0,09880 μg/ml, 0,008345 μg/ml and 0,004287 µg/ml, respectively. Anti-IFNAR1 antibody MDX-1333, 9D4WT and 9D4TM neutralized with leukocyte IFN (B) with IC50 values 1,121 mg/ml, 0,02104 μg/ml and 0,02120 µg/ml, respectively. Anti-IFNAR1 antibody MDX-1333, 9D4WT and 9D4TM neutralize IFNα 2b (B) with IC50 values 0,0006462 Anti-IFNAR1, 0,002789 Anti-IFNAR1 and 0,0008279 µg/ml, respectively. Anti-IFNAR1 antibody MDX-1333, 9D4WT and 9D4TM neutralize IFNω (G) with IC50 values 5,323 μg/ml, 0,01015 μg/ml and 0,01423 µg/ml, respectively. Anti-IFNAR1 antibody MDX-1333, 9D4WT and 9D4TM neutralized with IFNβ (D) with IC50 values 18,97 μg/ml, 0,7403 μg/ml and 0,2611 µg/ml, respectively.

Conclusion. These results show that the anti-IFNAR1 antibody MDX-1333, 9D4WT (unmodified) and 9D4TM (modified) exhibit similar specificity and neutralizing capacity for multiple type I interferons.

Example 24. Anti-IFNAR1 antibodies neutralize type I IFN in plasma of patients with SLE

Goal. To show the ability of anti-IFNAR1 antibodies to neutralize type I IFN in plasma obtained from the patients � SLE, using the measurements of reporter method.

Methods. Stably transfected cells PIL-5 ISRE maintained in medium RPMI 1640+1× penicillin-streptomycin-glutamine + 10% FTS and seeded with 100,000 cells per well in white/transparent 96-well plates Optilix (VWR, West Chester, PA). Titration of antibody is added to appropriate wells to final concentrations ranging from 90 μg/ml to about 60 PG/ml. serum Samples of patients with SLE, positive for interferon type I, is added to each well to a final serum concentration of 50% per well. Cells, and IFN antibodies were incubated over night at 37°C in an atmosphere of 5% CO2. After incubation overnight, the cells rapidly precipitated at 1200 rpm for 5 min and amplification of luciferase protein assessed by cell lysis reagent for lysing cells in Cell Culture Lysis and visualize, using the system of research luciferase Luciferase Assay System (firm Promega, Madison, Wi). The signal measure in imp./h and IC50 curves get, for analysis using the software GraphPad Prism 4.

Results. Antibody 9D4-TM neutralizes the type I interferons in the plasma of patients with SLE. The results of the study of neutralization of IFN type I in the plasma of patients with SLE are shown in Fig.24. Neutralization of interferon type I contained in the plasma sample of a patient with SLE, specifically ant is�body 9D4-TM compared to isotype control at higher antibody concentrations. Especially 9D4-TM takes the value of IC50 of 0.04 nm for the neutralization of IFN type I in the plasma sample taken from a patient with SLE.

Conclusion. This result indicates that the modified anti-IFNAR1 antibody 9D4-TM is able to effectively neutralize the type I interferon in patients with SLE.

Example 25. Anti-IFNAR antibodies inhibit induced IFNα population among MPC ciav

Goal. To show the ability of anti-IFNAR1 antibodies to inhibit the accumulation of MPC cells in the peripheral blood of the mouse model of SLE.

Methods. From mice used in the experiments in example 13, also take samples for analysis in the present example. PBMC isolated from spleen, lymph nodes, bone marrow and peripheral blood using standard methods of extraction and colored markers B220 and Ly6C on the surface of cells. Selected PBMC analyzed by FACS method and double-positive cells (B220 and Ly6C) assess the quality of MPC cells and related populations is shown in Fig.25.

Results. Fig.25 it was shown that ectopic expression of IFNα triggers the growth of MPC cells among PBMC, isolated from spleen (A), lymph nodes (B), peripheral blood (b) and bone marrow (D) in the presence of PBS or nonspecific murine IgG. Mice treated with anti-IFNAR antibodies, cells do not accumulate MPC in response to IFN-alpha. Mice treated with control �adenoviruses, do not accumulate MPC cells in the PBMC population.

Conclusion. These results show that anti-IFNAR antibodies specifically inhibit IFNa induced increased regulation of MPC cells.

Example 26. The modified anti-IFNAR1 antibodies exhibit reduced binding affinity to Fc receptors.

Goal. Estimation of the relative binding affinity of the modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM compared to the original unmodified antibody 9D4 with different Fc receptors.

Methods. All experiments were carried out on the instrument BIAcore 3000 (firm BIAcore, Inc., Uppsala, Sweden). In a typical experiment, 1 μm solutions of antibodies 9D4 IgG used for the immobilization of about ~7000 KE to ~11000 KE on the surface of sensor chip CM 5, using the standard connection Protocol of amino acids (company BIAcore, Inc.). Separately on each chip also get a clean surface, using the same Protocol, but without protein. Throughout the experiment such clean surfaces are used as cell control and provide for the correction of nonspecific binding and some artifacts of the 'household'. For the experiments on the binding tests are given to FcγRI in a concentration of 20 nm in buffer HBS-EP (firm BIAcore, Inc.), consisting of: 10 mm HEPES buffer, rn,4, 150 mm NaCl, 3 mm EDTA and 0.005% P20. Between injections FcγRI produce IgG on the surface of the ITB�m injection of 5 mmol of HCl, lasting 1 min. Overlap sensogram get using the software BIAevaluation 4.1 (firm BIAcore, Inc., Uppsala, Sweden).

Results. Anti-IFNAR1 antibody 9D4 and modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM can be tested for binding affinity to the immobilized protein FcγRI in the format BIAcore studies. Fig.26 shows that the anti-IFNAR1 antibody 9D4 exhibits a high affinity to immobilized FcγRI. Binding of anti-IFNAR1 antibodies 9D4 with FcγRI is specific, since a similar study with egg albumin has a very small affinity to the immobilized receptor. The modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM exhibit a reduced affinity of immobilized receptor FcγRI compared to the unmodified 9D4 anti-IFNAR1 antibody.

Conclusion. Received low affinity against FcγRI shown by the modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM, means that these antibodies may have reduced ability to activate ADCC in vivo.

Example 27. Fc receptors exhibit reduced binding affinity with modified anti-IFNAR1 antibodies.

Goal. To assess the relative binding affinity of various Fc receptors with modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM and the original non-modified anti-IFNAR1 antibody 9D4.

Methods. Measurement of surface plasma reason�NSA.

All experiments were carried out on the instrument BIAcore 3000 (firm BIAcore, Inc., Uppsala, Sweden). In a typical experiment, 1 μm solution of FcγRI used for immobilization of about ~7000 KE to ~11000 KE protein on the surfaces of the sensor chip CM 5, using a standard Protocol compound amino acid (BIAcore, Inc.). Separately on each chip also get a clean surface, using the same Protocol, but without protein. Throughout the experiment such clean surfaces are used as cell control and provide for the correction of nonspecific binding and some artifacts of the 'household'. For the experiments on the binding tests are given antibodies at a concentration of 333 nm in buffer HBS-EP (BIAcore, Inc., consisting of: 10 mm HEPES buffer, pH 7.4, 150 mm NaCl, 3 mm EDTA and 0.005% P20. Between injections FcγRI antibody produced IgG on the surface by injection of 3 M MgCl2lasting 1 min. Overlap sensogram get using the software BIAevaluation 4.1 (firm BIAcore, Inc., Uppsala, Sweden).

Results. Anti-IFNAR1 antibodies 9D4, 9D4-TM and 9D4-DM immobilized and incubated with soluble FcγRI. Binding affinity of soluble FcγRI receptor with each of the anti-IFNAR1 antibodies measured by BIAcore method, and the resulting curves are shown in Fig.27 A, B and C. the Receptor FcγRI binds immobilized anti-IFNAR1 antibody 9D4 with high affinity, as shown nafig.27A. This interaction is highly specific, as egg albumin does not show any binding to immobilized anti-IFNAR1 antibody 9D4. The modified antibodies 9D4-TM and 9D4-DM in contrast to the unmodified antibody 9D4 not bind FcγRI as much. Fig.27B modified anti-IFNAR1 antibody 9D4-DM immobilizer and or incubated with soluble FcγRI or egg albumin. Receptor FcγRI exhibits low binding affinity to the immobilized antibody 9D4-DM. Such binding affinity is similar to non-specific interaction observed in the case of soluble egg albumin. Fig.27V modified anti-IFNAR1 antibody 9D4-TM immobilizer and or incubated with soluble FcγRI, or with egg albumin. Receptor FcγRI exhibits low binding affinity to the immobilized antibody 9D4-TM. Such binding affinity is similar to non-specific interaction observed in the case of soluble egg albumin.

Conclusion. Low affinity exhibited by Fc receptor FcγRI against immobilized modified anti-IFNAR1 antibodies 9D4-DM and 9D4-TM relative to the unmodified anti-IFNAR1 antibodies 9D4 means that the modified antibodies may exhibit a reduced ability to induce the ADCC response.

Example 28. Anti-IFNAR antibodies block-dependent IFNα induction of genes

A�. To show the ability of anti-IFNAR antibodies block-dependent IFNα induction of genes in mice models of SLE.

Methods. From mice from the experimental procedures described in example 13, also receive samples for analysis in this example. After 8 weeks in the experiment, the mice were sacrificed and removed the kidney tissue. Not more than 50 mg of tissue used for RNA extraction using lysing buffer RLT (Qiagen company). The sample is placed in lysing buffer and lysing matrix A (firm Qbiogene) and treated for 30 sec at 4.5 m/sec, using a homogenizer Fastprep24 (company Thermo Electron Corporation, Waltham, mA). For RNA extraction thawed tissue lysates at first ProcessInput using spin Qiashredder column, then equal volumes of 70% ethanol was added to the tissue lysates and RNA purified using kits mini-kits for spin columns (Rneasy, following the manufacturer's instructions. From 3 μg of RNA get cDNA using reverse transcriptase Superscript III primer and oligo-d(T) according to the manufacturer's Protocol (firm Invitrogen, Corp. Carlsbad, CA). Samples of cDNA diluted in water, not containing restrictase, and stored at -80°C.

The levels of expression of selected genes measured by PCR real-time TaqMan® using the ABI 7900HT Fast Real-time PCR (firm Applied Biosystems, Foster City, CA). Gene β-actin related genes to "household" is used for e�degennaro control. The final volume of the reaction mixture to 20 μl and consists of 1 μl cDNA, 2 μl of 20× primer and probe (a method of gene expression TaqMan®, the firm Applied Biosystems) and 18 ml of the diluted mix TaqMan® Fast Universal PCR Master Mix. The conditions of amplification: 20 sec at 95°C, 50 cycles of 1 sec at 95°C and 20 sec at 60°C. the magnitude of the PT range from 0 to 50, and the last number indicates the lack of product formation. Quantification of gene expression is carried out using the CT comparative method (Sequence Detector User Bulletin 2; firm Applied Biosystems) and expressed as the ratio of the magnitude of the difference relative gene "household".

Results. Fig.28 shows the results of the comparative analysis of gene expression in the kidneys of 6 genes induced by interferon alpha, 8 weeks in mice models with enhanced manifestation of lupus. Mice expressing innate interferon alpha, treated with mouse IgG or anti-IFNAR antibodies. After 8 weeks in mice treated with control IgG, shown at a high level induction of genes responsible for IFNα, namely, ICAM1, VCAM1, CXCL9, CXCL10, and IFIT1. In mice treated with anti-IFNAR antibodies, not shown induction of genes responsible for IFNα, 8 weeks.

Conclusion. In mice models with enhanced manifestation of lupus, treatment with anti-IFNAR antibodies blocks the induction in the kidney of six genes (ICAM1, VCAM1, CXCL9, CXCL10 and IFIT1), mediated innate exp�esserman IFN-alpha compared to control mice by measuring the Taqman method. These results show that anti-IFNAR antibodies capable of blocking mediated IFNα signal transmission in the mouse model of SLE.

Example 29. Anti-IFNAR antibodies inhibit the accumulation of autoantibodies in serum

Goal. To show the ability of anti-IFNAR antibodies to inhibit the accumulation of autoantibodies in the serum of mice models of SLE.

Methods. From mice from the experimental procedures described in example 13, also receive samples for analysis in this example. Samples of whole blood taken from the interval 1 week 2 through week 7 the mode being used. Levels of serum anti-dndn antibodies determined by ELISA method. Briefly, tablets ELISA pre-treated poly(L-lysine) (100 µg/ml), cover with activated DNA of calf thymus (5 µg/ml in carbonate-bicarbonate buffer (SIGMA). After incubation over night at 4°C the plates blocked with PBS/10% FTS. Serum (dilution 1/200) was incubated for 30 min at room temperature. Bound IgG detected using conjugated with peroxidase goat anti-mouse IgG (1/4000) (KPL), added to the plates for 30 min. Binding was measured by addition of TMB substrate (KPL) and stop solution (KPL), and the optical density read at 450 nm. Standard murine anti-dndn IgG in serum serially diluted in each tablet (from 625 ng/ml) (firm Alfa Diagnostic) �La standardization.

Results. Fig.29 shows the results obtained by the method of ELISA, the levels of anti-dhank in the serum of mice during the course in mice models with enhanced manifestation of lupus. Mice, congenital expressing IFNα, treated with anti-IFNAR antibodies or control mouse IgG antibodies for 7 weeks. Mice not treated with anti-IFNAR antibodies, do not accumulate anti-dndn antibodies to the same extent or in the same extent compared with mice treated with control IgG antibodies. In mice infected with a control adenovirus did not develop anti-dndn antibodies during the course.

Conclusion. These results show that anti-IFNAR antibodies reduce the accumulation of anti-dndn antibodies in response to elevated levels of IFN-alpha.

Example 30. Anti-IFNAR antibodies reduced proteinuria in mice models with enhanced manifestation of lupus.

Goal. To show the ability of anti-IFNAR antibodies formed to reduce proteinuria (mounted therapeutically) on the model of mice with SLE.

Methods. From mice from the experimental procedures described in example 13, also receive samples for analysis in this example. However, when therapeutic approach in mice allow the development of proteinuria in the form of symptom of lupus prior to application of antibodies. Specifically allow the development of proteinuria in mice with a score of 2.0-2.5, described� earlier. When the threshold level of proteinuria has been completed, carry out the treatment regimen doses of PBS, control IgG or anti-IFNAR antibodies twice a week for 5 additional weeks. Twice a week take urine samples for analysis and assessment of proteinuria.

Results. Fig.30A presents the results of therapeutic studies of anti-IFNAR antibodies that reduce the evaluation of proteinuria in mice, representing a model with a higher manifestation of lupus. In short, it prevented the development of proteinuria mice and animals in different groups were injected or PBS or a control IgG, or anti-IFNAR antibodies as a treatment. From the data in the figure it follows that the assessment of proteinuria is reduced only in the group receiving anti-IFNAR antibodies. In mice receiving treatment FSB or control IgG, continues to increase over time to assess the degree of manifestation of proteinuria. (B) Analysis of area under the curve for the results obtained in five weeks, showed that the group in which mice were treated with anti-IFNAR antibody, different from both groups of mice treated with only PBS or a control IgG, and in which the results were similar.

Conclusion. These results show that anti-IFNAR antibodies can be used in therapeutic treatment of SLE.

Example 31. Anti-IFNAR antibodies reduce mortality in mice models with enhanced expression of the volcha�NCI.

Goal. To show the ability of anti-IFNAR antibodies to reduce the mortality of mice with established therapeutic model of SLE.

Methods. From mice from the experimental procedures described in example 13, also receive samples for analysis in this example. In therapeutic approach allowed the development in mice of proteinuria as a symptom of lupus before the application of antibodies. Specifically allow the development of proteinuria in mice with a score of 2.0 to 2.5, as described previously. When the threshold level of proteinuria has been completed, carry out the treatment doses of PBS, control IgG or anti-IFNAR antibodies twice a week for 5 additional weeks. The overall mortality rate trace for an additional 4 weeks.

Results. Fig.31 shows the mortality rates obtained from therapeutic studies of anti-IFNAR antibodies on the model with a reinforced manifestation of lupus. Briefly, after the formation of proteinuria in the group of mice injected or PBS or a control IgG, or anti-IFNAR antibodies as a treatment. In mice treated with anti-IFNAR antibodies, do not celebrate the deaths to 5 weeks, although in mice treated with the PBS or a control IgG, the degree to which mortality reaches of 87.5% and 62.5% respectively. In addition, during 9 weeks of the study, animals treated with anti-IFNAR antibodies, manifests a high degree of survival compared to now�governmental, processed PBS or a control IgG.

Conclusion. The results in this example demonstrate that the anti-IFNAR antibodies can reduce the mortality associated with lupus.

Example 32. No-mediated antibody 9D4-TM action ADCC.

Goal. To confirm that the antibody 9D4-TM can not induce ADCC activity due to its inherent weak binding affinity with receptors FcγRI and FcγRIIIA, conduct a series of experiments.

Methods. Target cells 293F mark label for cells DiO (firm Invitrogen, experiments I and II) and combine with unmarked PBMC effectors (for 4 h at 37°C) in the absence or presence of 10 μg/ml antibody 9D4-TM, isotype human IgG1 as a negative control R3-47, 9D4-WT or anti-EphA2 antibody used as positive control. Lysis of target cells is evaluated by measuring DiO+/PI+(reflects iodata) double positive staining. The ratio of effector-target is 50-1, the percentage of lysis calculated from the formula: [(percent of double positive staining in the presence of the antibody, the percentage of double positive staining only in the medium) / (the percentage of double positive staining in lysing buffer, the percentage of double positive staining only in the environment)]. One hundred percent lysis achieved by addition of lysing buffer (firm Promega.

In another embodiment, the target cells 293F incubated with the cell line transgenic NK cells stably expressing FcγRIIIA (experiment III) for 4 h at 37°C in the absence or in the presence of 10 μg/ml antibody 9D4-TM, isotype human IgG1 as a negative control R3-47, 9D4-WT or anti-EphA2 antibody used as positive control. The ratio of effector-target is 4-1, the percentage of lysis calculated from the formula: 100× (experimental value - effector spontaneous value - target spontaneous value) /(target maximum - target spontaneous value).

In experiments I and II (the ratio of ciav-N is 50-1) the percentage of lysis was calculated according to the formula; [(percentage of double positive staining in the presence of the antibody, the percentage of double positive staining only in the medium) / (the percentage of double positive staining in the presence of lysing buffer, the percentage of double positive staining only in the environment)]. In experiment III (the ratio of lines of transgenic NK cells expressing FcγIIIA to N is 4-1) the percentage of lysis was calculated according to the formula: 100× (experimental value - effector spontaneous value - target spontaneous value) /(target maximum - target spontaneous value).

Results. The modified antibody 9D4-TM or nomodifier�lized antibody 9D4-WT do not show a recorded action ADCC against cells 293F, exceeding the action in the case of antibodies R3-47 (PL.4). In contrast, the positive control antibody, anti-EphA2 antibody, causes a two-fold increase in cytotoxicity relative to the background level. These results confirm that the antibody 9D4-TM cannot mediate ADCC against targets expressing IFNAR1.

Table 5.
Evaluation of the effects of ADCC anti-IFNAR1 antibodies.
AntibodyExample I % lysis of the targetExample II % lysis of the targetExample III % lysis of the target
Positive control: anti-EphA233±436±143,4±0,5
Negative controls: R3-4714±118±318,1±1,1
9D4-WT14±220±217,5±1,6
9D-TM14±220±2Not tested

Conclusion. These results show�Ute, that the modified anti-IFNAR1 antibody 9D4-TM does not stimulate detectable actions ADCC directed to target cells expressing IFNAR1.

Example 33. Three-dimensional structure of the Fc region of a human, comprising mutations L234F/L235E/P331S.

Goal. To determine the three-dimensional structure of the Fc region human IgG1, including mutations L234F/L235E/P331S (Fc-TM).

Methods

Purification of Fc-TM. A fragment of the Fc/TM person is obtained by enzymatic cleavage of antibodies 9D4-TM. Cleavage was carried out using immobilized fitsin, according to the manufacturer's instructions (firm Pierce). The first cleaning is carried out on columns HiTrap protein a according to the manufacturer's instructions (GE Healthcare, Piscataway, NJ). After dialysis in 50 mm NaOAc/pH 5.2 solution of the protein applied to the column (HiTrap SP HP (GE Healthcare) and collect in excluded volume. Excluded volume loaded onto the column (HiTrap Q (GE Healthcare) and eluted in a gradient of NaCl to obtain a homogeneous drug Fc/TM, as measured by the method of SDS-PAGE in reducing and under non-reducing conditions. Fc-TM SDS-PAGE profile shows the presence of only one band of about 25 kDa or 50 kDa in reducing and under non-reducing conditions, respectively. This observation clearly indicates the presence of at least one disulfide bond within the chain on the status S and/or S. Consequently, mutant "below" the remains F234 and E contained in polypeptide� chain representing the crystal.

Crystallization of the protein is Fc-TM. Purified protein Fc-TM concentrated to approximately 5 mg/ml using a Centricon concentrator (firm Millipore, Billerica MA, cut off 30 KDa). Crystallization conditions of the identify using commercial screening from Hampton Research (firm Hampton Research, Aliso Viejo, CA), Emerald BioSystems (firm Emerald BioSystems, Inc., Bainbridge Island, Washington) and Molecular Dimensions (firm Molecular Dimensions Inc., Apopka, FL). Each screening gives several potentially suitable crystallization conditions. When optimizing get diffraction quality crystals of 0.2 M zinc acetate, 0.1 M imidazole malate, pH 8.0, 5% PEG 3350, 5% glycerol at a concentration of 2.0 mg protein/ml. In these conditions, well-formed crystals with three dimensions, varying from 0.1 to 0.2 mm, and grown for 2-3 days.

The collection of data. Data get diffraction from a single crystal in the Center for advanced research in biotechnology (Center for Advanced Research in Biotechnology - firm CARB, University of Maryland biotechnology Institute, Rockville, MD) using a rotating anode generator Rigaku MicroMax™ 007 with a signal plate R-AXIS IV++ (firm Rigaku/MSC, The Woodlands, TX). Before cooling, the crystal is kept for several minutes in the solution for growth with addition of 20% glycerol. Then the crystal was cooled to 105°K with a cryogenic cooler X - stream 2000 (Rigaku/MSC). Diffracted to 2 Å is achieved after one cycle of annealing according to the description Oganesyan, etc., 2007. Data on diffraction, including 234 image, harvested using a fluctuation range of 0.5°, the crystal/detector distance of 200 mm and an exposure time of 600 sec. Data pooled and scaled using the software HKL 2000 (Otwinowski &Minor, 1997).

The structure definition. Molecular replacement, structure refinement and calculation of the electron density was carried out using the software SSR (firm Collaborative Computational Project). C-face centered orthorhombic crystal contains 58% of the solvent and VM2,9, allowing one Fc polypeptide in the asymmetric unit cell. The crystal structure of the Fc/TM determined by molecular replacement and refinement in the resolution of 2.3 Å. The structure of the human Fc corresponding to PDB ID No 2DTQ (Matsumiya et, J. Mol. Biol. 368, 2007, cc.767-779), used as a model because of its high resolution and nelegantly condition. In particular, domains CH2 and CH3 were evaluated separately to minimize any bias in terms of the relative conformation of domains. Data to 3.0 Å is used for the purpose of molecular replacement, using Phaser (MSO, etc., Acta Cryst. D61, 2005, cc.458-464). After cleansing solutions end-LL-increment and Z-score are 1192 and 31, respectively. Weighted electron density, calculated using FWT/PWHT at 3.0 Å, shows a good comparison with the model except for some�'s loops in C H2 and CH3 domains. A strong positive difference electron density calculated using DELFWT/PHDELWT, see at the expected location of N-linked carbohydrate residues attached to N297. Any density is missing in any of the hinge residue before the residue at position 236, the result presumably refers to the high elasticity of this area. Found that only two previously described elegantnye Fc patterns people can identify regulations 234 and 235 (2DTQ/2DTS; Matsumiya, etc., J. Mol. Biol. 368, 2007, cc.767-779). Also cannot be visualized residues at positions 446 and 447. The residue at position 331 was first constructed in the form of alanine.

Several alternating cycles of refinement using the program Refmac 5" (Murshudov et, Acta Cryst. D53, 1997, cc.240-255) and design patterns manually using a graphical computer program "O" (Jones et al., (1991) Acta Cryst. A47, 110-119) converge with a value of Rfactorcomponent of 21.6, and the value of the free Rfactorgap of 27.5 for data in the resolution of 2.3 A. After the first cycle of refinement according to the electron density identified position sharidny residues and substitution of the serine residue at position 331. At other stages of refinement, the model was analyzed using the TLS Motion Determination (TLSMD) received from corresponding with�of rvera (Painter and others, J. Appl. Cryst. 39, 2006, cc.109-111; Painter and others, Acta Cryst. D62, 2006, cc.439-450). The following cycles of refinement were performed using the program method of clarifying rigid models on certain groups of five residues (residues 236-324, 325-341, 342-358, 359-403 and 404-445). Zinc ions present in the buffer solution for crystallization was identified by the electron density and determined their position in the model, if allowed coordination sphere and the distance. In particular set of coordinates one zinc ion for n and N. Another coordinate on N and N in symmetrically correlated the polypeptide. Two other zinc ion is located at the coordinates (E-318 and E. In all cases, water molecules complementary to the expected coordination sphere of zinc ions. The carbohydrate part of the molecule is modeled in the electron density, and the final model contains nine sugar residues, essentially we described in the context of another Fc patterns (Oganesyan, etc., Molecular Immunology, December 11, 2007, in press). The final model contains 75 of the solvent molecules. Data of crystallography and cleaning statistics lead in the table.6.

Table 6.
Collecting x-ray diffraction data and refinement statistics of the model.
Wavelength (Å) A 1.54
Resolution (Å)36,83-2,30 (2,38-2,30)and
Space groupC2221
The unit cell parameters (Å)50,18, 147,30, 75,47

The total number of reflexes54,409
Unique reflexes12,617
Average redundancy4,31 (2,72)a
Integrity (%)98,3 (90,0)and
X-the set of intensities (Rmerge)0,062 (0,300)and
I/σ (I)13,0 (3,3)and
R factor/Free R factor0,216/0,275
The standard deviation is isomorphic sets (RMSD bonds, Å0,012
The standard deviation is isomorphic sets (RMSD) angles (°)1,48
Residues in most before�occhialini region {φ,ψ} space (%) 89,9
Residues in additionally allowed region {φ,ψ} space (%)10,1
The number of atoms in the protein1678
The number of atoms that are not part of a protein189
In factor (model/Wilson), Å243/40
andThe values in brackets correspond to the shell of highest resolution.

Results. Fc-TM crystallizes in the space group C2221one polypeptide in the asymmetric region (Fig, 32). Get the diffraction pattern of a crystal with a resolution of 2.3 Å and with a relatively high average mosaicity of 1.26°. This high mosaicity due to the properties of cold, crystals and non-refrigerated. All the residues in positions 236-445 can be traced in the electron density, and do not observe electron density for residues of the hinge region in front of the position 236 to compensate for the invisibility of mutations L234F and L235E. Electron density at position 331 corresponds to serina.

Atomic coordinates and experimental structure factors Fc-TM have been deposited in protein data Bank Protein Data Bank under but�'erom in the directory 3C2S.

The overall three-dimensional structure of Fc-TM very close to the previously described structures elegantnyh areas Fc (Deisenhofer, Biochemistry, 20, 1981, cc.2361-2370; Krapp et, J. Mol. Biol. 325, 2003, cc.979-989; Matsumiya, etc., J. Mol. Biol. 368, 2007, cc.767-779; Oganesyan, etc., Molecular Immunology, December 11, 2007, in press). More precisely the structure of the human Fc match PDB ID numbers 1H3W (Krapp et, J. Mol. Biol. 325, 2003, cc.979-989) and 2QL1 (Oganesyan, etc., Molecular Immunology, December 11, 2007, in press), which is closest to Fc-TM in respect of unit cell parameters, the content of the asymmetric field, space group and packing. In individual assessment domains of Fc-TM CH2 and CH3 are characterized by high rigidity and stiffness of the structure when compared with other elegantnymi nematandani structures of the human Fc. For example, RMS coordinating the displacement of atoms From, are 0.6 and 0.4 Å for domains CH2 and CH3, respectively, after the application of Fc-TM chain And PDB ID number 2DTQ (Matsumiya et, J. Mol. Biol. 368, 2007, cc.767-779).

Table.7, below, provides the coordinates of the atomic structure of the antibody Fc-TM. In table.7 presents the following abbreviations.

Designation of type "atom" refers to the element whose coordinates are provided. The first letter in the column means the item.

The symbol "AA" means amino acid.

Notation "X, Y, and Z denote the Cartesian coordinates of the element.

"In" means� the temperature factor, which corresponds to the movement of the atom around its atomic center.

"OSS" refers to employment and represents the percentage of time during which the atom takes a certain type in specific coordinates. The magnitude of the SIS range from 0 to 1, where 1 corresponds to 100%.

Conclusion. It is established that the three-dimensional structure of the Fc/TM is very similar to the structure of other elegantnyh neutropenic Fc regions of a human. Expressed widely varying functional effects of a set of TM substitutions were not caused major structural rearrangements in the structure of the Fc, and is preferable due to the localized loss of several interactions at the sites of mutations.

Example 34. Internalization of anti-IFNAR1 antibodies

Goal. The study of the anti-IFNAR1 antibody internalization in cells.

Methods. Cells THP-1 were cultured in RPMI-1640 medium containing 0.05 mm 2-mercaptoethanol and 10% fetal SIV�rocky calf at 37°C in an incubator with 5% CO 2. Cells THP-1 was seeded at 2×105cells/ml in fresh culture medium the day before the experiment. On the day of experiment, cells washed, counted and re-suspended in the FSB in the amount of 3×106cells/ml Cells stained with 1 μm CFSE at 37°C in an incubator with CO2within 10 min. After two additional washes in PBS, the cells were placed on ice and incubated with FcR blocker, using 20 µl of 106cells on ice for 5 min, and then stained with 1 μg/ml Alexa647-9D4-TM or Alexa 647-R347 (non-specific control antibody) on ice for 1 h. After removal of unbound mAb by 3 washes of PBS cells was resuspended in PBS containing 2% BSA and sodium azide. Begin the internalization by transferring cells into the camera with the control environment: a temperature of 37°C, 5% CO2and 70% humidity, and the kinetics of internalization of Alexa647-9D4-TM record over time of fluorescence observation cells.

The fluorescence of the cells are analyzed using the algorithm. In this algorithm, apply the cytosolic dye CFSE to identify communication cells and membrane area. Using the count algorithm associated with the antibody 9D4-TM fluorescence inside the cells and on the membrane. The degree of fluorescence accumulated inside the cells, counted using a model fitting data using about�program ensuring SAAMII.

Results. Alexa647-9D4-TM binds to cells THP-1. Do not watch the binding of Alexa647-R347, isotype control 9D4-TM, on the same cells. This result shows specific binding to cells THP-1 9D4-TM (Fig.33). At 4°C binding of 9D4-TM is predominantly localized on the cell surface (0 min - Fig.33). If the cells were incubated at 37°C the fluorescence signal for staining 9D4-TM was significantly downgraded from the cell surface and accumulates in the cytosolic compartment in the form of discontinuous spots. Kinetic images recorded for 60 min, show a gradual migration of the fluorescence from the cell surface to the intermittent spots localized in the cytosolic compartment (15, 30 and 50 min, Fig.33). The results clearly show the internalization of antibodies 9D4-TM cells, THP-1.

Example 35. Lack of action CDC, mediated by antibody 9D4-TM

Goal. Conduct a series of experiments to determine whether the antibody 9D4-TM to induce action by the CDC.

Methods. Freshly drawn blood (about 100 ml) from healthy human donors were collected and pelleted by centrifugation for 10 min at 3000 g to separate the serum from the cells. The serum fraction is separated into two tubes. The contents of the first vial is diluted with RPMI medium 1640 without phenol to a final concentration of 10% serum (not inactivated by heating (NEITHER�)). The second tube is then placed in a water bath to 56°C and kept for 30 min for heat inactivation of components of complement. Then, the contents of the second vial is diluted with RPMI medium 1640 without phenol to a final concentration of 10% human serum, inactivated by heating (INF).

Cells In Daudi used as target cells because they Express CD20 (the target for the antibody positive control) and IFNAR1. Target cells are washed and was resuspended in RPMI medium without phenol with 10% inactivated by heating the serum or in medium RPMI without phenol with 10% inactivated by heating serum to a final concentration of 0.4×106cells/ml Solutions of antibodies is obtained in the form of a 3× dilution series with concentrations ranging from 50 μg/ml to 1.3×10-6µg/ml Repeatable solutions of preparations of antibodies produced in each environment - and with heat inactivated human serum, and with mainactivity warm human serum. The CDC study is produced by adding 50 µl of media or YING ning in the corresponding round-bottomed wells of 96-hole tablet. In appropriate wells add 50 µl of serial solutions of antibodies. Then in the wells contribute 50 µl of drugs target cells, including additional wells only with target cells as a control. The plates were incubated at 37°C for 4 h in an atmosphere of 5% With� 2. After 3.5 h incubation add 20 ál lytic buffer in corresponding control wells that are designed to determine the maximum signal lysis. Analysis of the release of lactate dehydrogenase (LDH) Quantitate™ is carried out using protocols described in the study of non-radioactive cytotoxicity of the company Promega No. 01780. The absorption was measured at 490 nm, and a Kd value get using software for analysis GraphPad Prism 4.

Results. Fig.34 presents the results according to the CDC, obtained as explained above. The modified anti-IFNAR1 antibody 9D4-TM does not exhibit detectable actions CDC on target cells Daudi In excess of the same effect of the antibody R347. In contrast, a positive control antibody that binds to the CD20 antigen, expressed on Daudi cells, causes a dose-dependent increase in cytotoxicity, exceeding the background level. These results confirm that the antibody 9D4-TM is not able to mediate CDC in target cells expressing IFNAR1.

Example 36. The modified anti-IFNAR1 antibody 9D4-TM does not show any side toxicity

Goal. To confirm that the antibody 9D4-TM does not cause any side toxicity study the toxicity caused by a single dose in macaques of Griboedov.

Methods. In the present study in 4 groups with 10 animals each�Aya (5/sex/group), animals receive a single dose of 0, 5, 30 or 100 mg/kg of antibody 9D4-TM on the first day. After dosing of 2 animals/sex/group were sacrificed and autopsied at 3 days, and all remaining animals subject to monitoring to 70 days and then removed from the study without opening. Toxicity was assessed by mortality, clinical signs (including menstruation), immunophenotyping, body weight, state of health (heart rate, respiratory rate and body temperature), clinical pathology, organ weights and microscopy data.

Results. In the conditions described above, there is associated with the antibody 9D4-TM unwanted changes in mortality, clinical signs (including menstruation), body weight, health status (heart beat, breathing rate and body temperature), clinical pathology, organ weights and microscopy data. These results indicate that the modified anti-IFNAR1 antibody 9D4-TM does not cause any adverse toxicity.

As specific embodiments of the present invention is shown for clarification of the description, specialists in this field it is obvious that numerous variations of the details may be made without departing from the scope of the scope of the present invention defined in the attached claims.

All publications, patents and patent�e applications referred to in the description of the present invention, included in the form of links, depending on how specifically each individual publication, patent and patent application, it is advisable to include in the present invention by reference.

1. Modified monoclonal antibody of class IgG specific against IFNAR1, wherein the said antibody comprises in the Fc region of human IgGl amino acid replacement L234F, L235E, and P331S, the numbering of which is given according to the EU index, developed by Kabat, and the antibody exhibits reduced affinity for Fcgamma RI, Fcgamma RIII and c1q receptors compared with unmodified antibody.

2. The antibody according to claim 1, wherein the said antibody comprises:
and. variable region CDR1 light chain that includes Seq ID NO: 1;
b. variable region light chain CDR2 that includes Seq ID NO: 2;
V. variable region CDR3 light chain that includes Seq ID NO: 3;
G. variable region CDR1 of the heavy chain that includes Seq ID NO: 4;
D. variable region CDR2 of the heavy chain that includes Seq ID NO: 5; and
E. variable region CDR3 of the heavy chain that includes Seq ID NO: 6.

3. The antibody according to claim 1, wherein the said antibody comprises:
and. variable region CDR1 light chain that includes Seq ID NO: 21;
b. variable region light chain CDR2 that includes Seq ID NO: 22;
V. VA�abelow region CDR3 light chain human comprising Seq ID NO: 23;
G. variable region CDR1 of the heavy chain that includes Seq ID NO: 24;
D. variable region CDR2 of the heavy chain that includes Seq ID NO: 25; and
E. variable region CDR3 of the heavy chain that includes Seq ID NO: 26.

4. The antibody according to claim 1, wherein the said antibody comprises:
and. the variable region of the heavy chain of a person, containing the amino acid sequence of Seq ID No: 38; and
b. variable region light chain of a person, containing the amino acid sequence of Seq ID No: 40.

5. The antibody according to claim 1, wherein the said antibody comprises:
and. the variable region of the heavy chain of a person, containing the amino acid sequence of Seq ID No: 18; and
b. variable region light chain of a person, containing the amino acid sequence of Seq ID No: 20.

6. The antibody according to claim 1, wherein the said antibody comprises:
and. the variable region of the heavy chain of a person, containing the amino acid sequence of Seq ID No: 28; and
b. variable region light chain of a person, containing the amino acid sequence of Seq ID No: 30.

7. Isolated nucleic acid providing for expression of an antibody according to one of the preceding paragraphs and comprising a polynucleotide sequence that encodes it.

8. Pharmaceutical composition for prevention, treatment and maintenance �whether the relief of diseases or disorders, mediated by interferon I, comprising a therapeutically effective amount of an antibody according to one of claims. 1-6 and pharmaceutically acceptable excipient.

9. Pharmaceutical composition according to claim 8 for use in the treatment of a disease or disorder that is selected from the group including: graves ' disease, Hashimoto's thyroiditis, Crohn's disease, psoriasis, psoriatic arthritis, sympathetic ophthalmic, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome, sjögren syndrome, scleroderma, Addison's disease, polyendocrine syndrome of deficiency, Guillain-Barre syndrome, immune thrombocytopenic purple, pernicious anemia, myasthenia gravis, primary biliary cirrhosis, mixed connective tissue disease, vitiligo, autoimmune uveitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, celiac disease, dermatitis herpetiformis, autoimmune hepatitis, pemphigus, pemphigus vulgaris, pemphigus foliaceous, bullous pemphigoid, autoimmune myocarditis, autoimmune vasculitis, alopecia alopecia, autoimmune atherosclerosis, Behcet's disease, autoimmune myelopathy, autoimmune hemophilia, autoimmune interstitial cystitis, autoimmune diabetes insipidus, autoimmune endometriosis, recurrent Paul�the chondrite, ankylosing spondylitis, autoimmune urticaria, dermatomyositis, syndrome of Miller Fisher, IgA nephropathy, goodpasture's syndrome and herpes pregnant.



 

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9 cl, 4 dwg, 5 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.

EFFECT: present invention can find further application in cancer therapy.

16 cl, 27 ex, 18 tbl

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.

EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.

4 cl, 3 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.

EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.

16 cl, 7 dwg, 13 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.

EFFECT: invention can find further application in diagnosing and treating cancer.

43 cl, 98 dwg, 20 tbl, 26 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunology. Presented are anti-Dickkopf 1 (anti-Dkk-1) antibodies and their functional fragments specified among the antibodies: 1) containing CDR1 VH containing the amino acid sequence SSYAIS, SYAIS or GFTFSSY; CDR2 VH containing the amino acid sequence SVSGTGLGFGTYYPDSVKG or SVSGTGLGFGTY; and CDR3 VH, containing the amino acid sequence TSLENYAFDY or SLENYAFDY; and CDR1 VL containing the amino acid sequence RASESVDDFGISFIN; CDR2 VL containing the amino acid sequence AGSKQGS; and CDR3 VL containing the amino acid sequence QQLKEVPPT; and 2) the antibodies disclosed in Table 4 presented in the application materials. Described are: nucleic acids coding the above antibodies or their functional fragments; expression vectors containing the above nucleic acids; and cells used for expression of the above antibodies or their functional fragments and containing the above expression vectors. Presented is a method for producing the antibody or its functional fragment involving the stage of culturing the above expression cell. Disclosed is a composition possessing Dkk-1 binding activity, containing the antibody or its functional fragment in a therapeutically effective amount and a pharmaceutically acceptable excipient, thinner or carrier.

EFFECT: invention enables extending the range of products for treating the diseases associated with Dkk-1 and LRP5/6 excessive reaction, which cause Wnt activation.

14 cl, 14 dwg, 14 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to method of obtaining bivalent bispecific antibody, which includes transformation of host cell by vectors, containing molecules of nucleic acids, coding first light chain and first heavy chain of bivalent bispecific antibody, and vectors, containing molecules of nucleic acids, coding second light chain and second heavy chain of bivalent bispecific antibody, cultivation of host cell under conditions, providing synthesis of molecule of bivalent bispecific antibody from said culture. Said antibody contains first light chain and first heavy chain of antibody, specifically binding with first antigen, and second light chain and second heavy chain of antibody, specifically binding with second antigen, in which variable domains VL and VH of second light chain and second heavy chain are replaced by each other and constant domains CL and CH1 of second light chain and second heavy chain are replaced by each other.

EFFECT: invention makes it possible to increase output of correct bispecific antibody by increasing the level of correct heterodimerisation of heavy chains of wild type and modification of heavy chains resulting from crossing over.

2 cl, 31 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a polypeptide containing two binding fragments presented by antibodies; the first of them binds to CD3e(epsilon) chain epitope of a human or a primate, other than a chimpanzee, particularly Callithrix jacchus, Saguinus oedipus and Saimiri sciureus; the second one - to EGFR, Her2/neu or IgE of a human or a primate, other than a chimpanzee, with the above CD3e epitope containing the amino acid sequence Gln-Asp-Gly-Asn-Glu. There are also disclosed a coding sequence of the nucleic acid, a vector, a host cell and a method for preparing the above peptide, as well as a pharmaceutical composition and using the polypeptide in preventing, treating or relieving a proliferative disease, a malignant disease or an immunological disorder.

EFFECT: invention provides the clinical improvement of T-cell redistribution and the enhanced safety profile.

17 cl, 8 tbl, 26 dwg, 26 ex

FIELD: biotechnology.

SUBSTANCE: bispecific antibody is proposed, that binds to both the blood coagulation factor IX/activated blood coagulation factor IX and with the blood coagulation factor X, and functionally replaces the function of blood coagulation factor VIII. The nucleic acid is considered, encoding the antibody of the invention, a vector, a cell and a method of producing the antibody, and also a pharmaceutical composition and a kit for use in the method of preventing and/or treating bleeding or diseases associated with or caused by bleeding.

EFFECT: invention may find further application in the treatment of diseases associated with impaired blood clotting.

16 cl, 2 ex, 6 dwg

FIELD: chemistry.

SUBSTANCE: claimed is bispecific antibody, which is bound with both blood coagulation factor IX/activated blood coagulation factor IX and with blood coagulation factor X and functionally replaced function of blood coagulation factor VIII. Described are nucleic acid, coding antibody by invention, vector, cell and method of obtaining antibody, as well as pharmaceutical composition and set for application in method of prevention and/or treatment of bleeding or diseases, associated with or induced by bleeding.

EFFECT: invention can be applied in therapy of diseases, associated with blood coagulation disorders.

16 cl, 2 ex, 6 dwg

FIELD: medicine.

SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.

EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.

18 cl, 124 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology. There are presented versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a specified amino acid sequence and at least one free cysteine amino acid residue specified in A118C (according to the European Numeration) in the heavy chain and V205C (according to the Kabat numeration) in the light chain. There are disclosed: versions of a conjugate compound of the antibody and a drug preparation, wherein the antibody is bond to the drug preparation through free cysteine; an antibody-based pharmaceutical compound for treating cancer; method for detecting CD79b or cancer cells, as well as a method for inhibiting cell proliferation using the conjugate compound. What is described is a method for producing the conjugate compound.

EFFECT: invention can find further application in the therapy of CD79b-associated cancer diseases, including treating haemopoietic tumours in mammals.

70 cl, 20 tbl, 9 ex, 51 dwg

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